The control of microtubule assembly in vivo.

INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 59

The Control of Microtubule Assembly in Vivo ELIZABETH C. RAFF Program in Molecular, Cellular, and Developmental Biology and Department of Biology, Indiana University. Bloomington. Indiana

1. The Secret: Observations of Microtubule Assembly in Vivo.

The Importance of Microtubule-Organizing Centers . . . . . A. The Morphogenesis of Basal Bodies and Centrioles . . . . B. The Assembly of the Axoneme of Cilia and Flagella . . . . C. The Morphogenesis of Microtubule-Containing Organelles in Protozoa . . . . . . . . . . . . . . . . . . . . D. The Assembly and Disassembly of Labile Microtubule Arrays: The Mitotic Apparatus . . . . . . . . . . . . . . . E. The Assembly and Disassembly of Labile Microtubule Arrays: Cytoplasmic Microtubules . . . . . . . . . . . . . 11. The Dance: Experimental Dissections of Microtubule Assembly in Vivo . . . . . . . . . . . . . . . . . . . . . . . A. The Biochemistry of Tubulin, the Structural Microtubule Protein B. Microtubule Assembly in Vitro: Possible Regulatory Factors . C. The Growth of Microtubules in Vitro onto Isolated Microtubule-Organizing Centers . . . . . . . . . . . D. Experiments in Which Microtubule Assembly Is Elicited in Vivo by the Injection of Microtubule-Organizing Centers into Eggs E. Calcium and Other Small Molecules as Possible Regulators of Microtubule Assembly in Vivo . . . . . . . . . . . . F. Time-Dependent Properties of Tubulin and Microtubules . . G . Spatial Localization of Microtubules: Association with Membranes . . . . . . . . . . . . . . . . . . . H. Microtubule Regulation: Genetic Studies . . . . . . . . 111. The Suppositions: Conclusions about the Control of Microtubule Assembly in Vivo . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . .

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40 51 58 64 69 73 16 81 83

We dance round in a ring and suppose, But the secret sits in the middle and knows. Robert Frost ( 1 949)

With the adv nt of electron microscopy, it was recognized that microtubules were ubiquitous components of eukaryotic cell organelles and were in fact participants in many of the most basic cellular processes, most notably as the spindle Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-364359-7

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ELIZABETH C. RAFF

fibers of the mitotic apparatus and as the 9 + 2 axoneme tubules in cilia and flagella. Somewhat later they were found to be one of the main components of neural tissue, and it is now known that microtubule networks exist in the cytoplasm of most eukaryotic cells. The exquisite and delicate control over the timing of appearance and positioning of microtubules and microtubule-containing organelles is spectacularly obvious in many cellular events, and the question of the nature of their regulation is thus a fascinating problem. But it is also a particularly frustrating problem because of the abundance of tantalizing data which approach the question but fall short of answering it. The secret, in this case, is all too elusive. The vastness of the microtubule literature and the frequency with which it is reviewed are often noted [generally, as here, at the beginning of the very reviews in question: for example, see Porter (1966) for the first review of microtubule function, and Burnside (1975) for a more recent historical overview; reviews by Hepler and Palevitz (1974), Jacobs and Cavalier-Smith (1977), Newcomb (1969), Olmsted and Borisy (1973), Pickett-Heaps (1975b), Roberts (1974), Snyder and McIntosh (1976), and Stephens and Edds (1976) provide several viewpoints on microtubule structure and function in animals and plants.] What follows therefore is in no way a comprehensive review of the microtubule literature or even of that part of it which might be supposed to be included under the title. The representation of papers discussed is just that, a selective representation. In 1974 Roberts summed up rather nicely what was then known about the control of microtubule assembly in vivo by noting that it was “all rather hazy at the moment.” Unfortunately, it is still rather hazy, but in the last several years much more information has been accumulated which both gives direct clues about the molecular mechanisms underlying the temporal and spatial regulation of microtubule assembly and makes the interpretations of some earlier observations (particularly on the role of microtubule-organizing centers, for example) more sound. This article, then, addresses the following questions. First, what exactly do microscopists see when they look at a cell in which microtubules are in the process of coming or going? Second, what kind of experiments are possible to examine microtubule assembly events in living cells? And third, what molecules in addition to the primary constituent of microtubules, tubulin, are involved in controlling these events?

I. The Secret: Observations of Microtubule Assembly in V i v a The Importance of Microtubule-Organizing Centers One of the surprises of the close look at cells allowed by electron microscopy was not only the ubiquity of microtubules but also the fact that very disparate structures were formed from them. For instance, the two most prevalent

CONTROL OF MICROTUBULE ASSEMBLY

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microtubule-containing organelles, mitotic spindles and cilia and flagella, are very different in that the first is a labile structure repeatedly and rapidly assembled and disassembled at each cell division, while the others are stable structures with diverse accessory structural components, persistent under treatments such as cold, pressure, and various mitotic poisons (e.g, colchicine) which cause the mitotic spindle to disappear. The question of how the precise regulation of both the temporal appearance and spatial organization of microtubules is achieved immediately became the subject of an enormous amount of research. Crucial to this question is the point of origin of microtubules, and investigation of this revealed yet another microtubule-containing organelle. Light microscopy showed dense structures at the poles of many mitotic spindles and at the base of cilia and flagella-the centriole and the basal body, respectively. Electron microscope studies showed these two structures to be morphologically equivalent, consisting of a cylindrical structure with a basic pattern of nine triplet tubules arranged around a central “cartwheel” structure [see Fulton (1971) and Pitelka (1974) for historical and morphological reviews of these structures]. However, although the outer doublet tubules of the axoneme are direct extensions of the basal body triplet tubules, the microtubules in the mitotic apparatus are not continuous with centriole tubules, and many mitotic spindles, notably those in higher plants, have no centrioles at the poles at all. The polar mitotic microtubules in fact appear to arise out of amorphous electron-dense material which surrounds the centriole (or, particularly in anastral mitotic figures, the polar tubules appear, like the earth in Genesis, to arise out of nothing at all). Furthermore, in addition to the highly structured basal body and the seemingly structureless pericentriolar material there are diverse other structures from which microtubules arise. The useful term “microtubule-organizing center” was coined by Pickett-Heaps (1969) to denote the structures or material from which microtubules initiate. A. THEMORPHOGENESIS OF BASAL BODIES A N D CENTRIOLES

Some of the most elegant descriptions of the process of microtubule assembly in vivo are the original observations of the formation of centrioles and basal bodies which were made as soon as the technology of electron microscopy permitted. These observations have stood in the literature awaiting complete interpretation from biochemical data (but the biochemical technology in this area has not quite caught up yet). Early on, centrioles were postulated to be selfreplicating organelles; the fact that new centrioles often arose close to mature ones, the difficulty of discerning intermediate forms, and the complexity of the organelle all seemed to indicate that they must be autonomous-‘ ‘reproducing” by dividing-r at least that the foramtion of a new centriole required the presence of a mature one and was directed by it. Even after it was realized that they

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could in fact arise de novo (Dirksen, 1961), the idea persisted. This problem has been discussed at length by Pickett-Heaps (1969, 1971, 1975a). In fact, centrioles and basal bldies in vivo arise out of electron-dense material of uncertain structure and composition, which may or may not be associated with a mature organelle. Studies on basal body and centriole morphogenesis in various vertebrate tissues have yielded similar reconstructions of the development of the mature organelle from precursor structures which are in turn derived from amorphous or fibrous electron-dense masses in the cell interior. Dirksen and Crocker (1966) examined the formation of centrioles in differentiating ciliated cells of fetal rat tracheal epithelium. Subsequently, Dirksen (197 I ) described centriole morphogenesis in the ciliated epithelium of mouse oviduct, which took place in a brief period after birth but was not synchronized, even within a single cell. Centriole morphogenesis was similar in both tissues, proceeding through four sequential stages. First, in the center of the cell clusters of electron-dense fibrillar masses 60-80 nm in diameter appeared, which later became organized into larger aggregates 100-700 nm in diameter in cleared areas of the cytoplasm. The structure of these aggregates was difficult to ascertain because of their electron opacity; they were usually amorphous, but occasionally microtubules or a mature centriole was present. As shown in Fig. 1 , these electron-dense masses then gave rise to procentrioles, becoming surrounded by as many as nine immature centrioles in various stages of development. The first stage in centriole development was the appearance of an annulus or disk of indistinct structure which later developed the typical centriolar cross section. The microtubules of the procentrioles were often connected to the central mass by fine strands. Concomitantly with centriole maturation the central mass of material either disappeared or became clearly hollow, suggesting that the centrioles had in fact been formed from this material. Occasionally procentrioles were found surrounding mature centrioles. In neither of these studies was Dirksen able to define clearly the relationship between mature centrioles and the amorphous material which appeared to be the earliest procentriole precursor. Steinman (1968) followed the differentiation of ciliated cells in epidermis and trachea of Xenopus faevis. His electron micrographs show dense, amorphous masses around which are clustered smaller electron-dense masses which apparently become procentrioles, cylinders 150 nm in diameter with nine single tubules. These structures appeared deep in the cytoplasm of the cell near the nucleus; correlated with their disappearance multiple mature centrioles 200 nm in diameter with a typical cross-sectional structure appeared in the apical cytoplasm. The inference was that the procentrioles rapidly matured and migrated to the cell surface, although no intermediate forms were found. The centrioles then aligned at the apical surface, ciliary shafts grew out of them, and basal body accessory structures such as rootlets appeared. Small, electron-dense bodies close to the base of the basal body and the growing cilium were interpreted as


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Flc. I . Centriole morphogenesis in developing mouse oviduct epithelium. Three centriole generative complexes, each consisting of the central electron-dense precursor structure (clearly “hollow” at this stage of development) surrounded by nine developing procentrioles. Two procentrioles in the upper right complex were not in the plane of section. X47.450. Reprinted from Dirksen (1971), with permission.

axoneme precursors; similar but less electron-dense material was interpreted as the source of the basal body rootlets. The various morphological events described did not occur in synchrony; a single cell often contained both basal bodies with growing axonemes and unaligned centrioles still in the supranuclear cytoplasm. Attachment of the centriole at the cell surface appeared to be the signal for assembly of the axoneme; Steinman saw only two cases in which an unaligned centriole bore an axoneme. A similar pattern of centriole formation was observed by Kalnins and Porter (1969)during ciliagenesis in chick tracheal epithelium. Basal bodies formed in a

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ELIZABETH C . R.AFF

region of fibrous material associated with the cell centrioles; procentrioles arose in a cluster around a core of dense material sometimes containing a cylindrical structure resembling a centriole. The earliest procentrioles were short cylindrical structures without microtubules; tubules appeared first as singlets, progressing to doublets and triplets. The basal bodies then elongated, matured, and migrated to the cell surface, where the cilia were assembled. In this tissue basal body development appeared to be synchronous. Sorokin (1968) studied the formation of centrioles and subsequent ciliagenesis in fetal rat lungs. He observed two patterns of morphogenesis. The first occurred early in development in interphase pulmonary cells of all types and involved the centrioles subsequently associated with the mitotic spindles. Usually one, but sometimes as many as eight, new centrioles arose directly adjacent (at right angles) to the wall of a preexisting centriole, first as procentrioles; during development these annular structures lengthened into cylinders. The triplet tubules started as singlet tubules to which the second and third tubule walls were added. These daughter centrioles were released into the cyoplasm when they had matured about halfway. An interesting and somewhat puzzling aspect of the regulation exerted in this system is Sorokin’s observation of the occasional growth of transitory rudimentary cilia from one of the pair of centrioles in differentiating fetal pulmonary cells. These are transitory embryonic organelles and are rare in adult tissues; they are distinguishable from adult cilia because they are incompletely formed, especially at the tips, and the central pair of tubules is lacking. It is tempting to interpret this observation as a lapse in control, that is, a centriole growing a cilium. The second pattern of centriole morphogenesis Sorokin observed was the acentriolar pathway, which occurred late in the fetal period and involved formation of the basal bodies of ciliated epithelial cells. Masses of fibrous, granular material accumulated in close proximity to Golgi elements in the apical cytoplasm. The fibrogranular areas increased in size and apparently condensed into spherical masses, around the periphery of which procentrioles arose. Ultimately the mature centrioles aligned in rows underneath the apical end of the cell membrane. The signal for ciliagenesis appeared to be placement of the basal bodies at the cell membrane; accessory structures such as satellites and roots appeared, and then the ciliary shaft grew out. The ciliated border cilia grew faster than the rudimentary cilia and had the complete 9 + 2 cross-sectional pattern. The mature basal bodies did not ususally have procentrioles associated with them, but Sorokin observed this in a few cases and concluded that the capacity for formation of an associated organelle is retained in basal bodies produced by the acentriolar pathway. A similar dual set of pathways for centriole morphogenesis was observed by Anderson and Brenner (1971) in rhesus monkey oviduct. After ovariectomy, deciliation and loss of basal bodies occurred in ciliated epithelial cells of the


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oviduct; estrogen treatment caused redifferentiation of these cells. The major pathway was acentriolar, in which basal bodies were formed from procentrioles generated from aggregates of fibers with no structural resemblance to centrioles. First, fibrous granules of 40-60 nm in diameter appeared in the apex of future ciliated cells. These granules usually were aggregated into sheets or spheres but sometimes were dispersed in the cytoplasm and sometimes associated with the nuclear membrane; occasionally a few microtubules were present among them. The fibrous material appeared to fuse to form the procentriolar ring. These investigators found the stages in procentriole development difficult to interpret. The central cartwheel structure formed before the triplet tubules; the A tubules then formed in sequence around the ring, but tubule growth after that was not synchronous. The transition from procentriole to basal body involved lengthening of the procentriolar cylinder and migration to the cell surface, followed by the addition of accessory structures and changes in the internal cartwheel arrangement. The ciliary shaft was assembled after the basal body reached the cell surface. The second, minor, pathway Anderson and Brenner observed was the centriolar pattern, in which 1 to 10 procentrioles formed at right angles to walls of the preexisting pair of centrioles. As in the acentriolar pathway, these procentrioles also appeared to form from amorphous electron-dense material which in this case surrounded the walls of the mature centrioles and in which their bases were embedded. The maturation pattern of these centrioles was the same as the acentriolar pathway; mature centrioles were released into the cytoplasm and apparently migrated to the surface along with those formed through the acentriolar pathway. Since all procentrioles associated with any one mature centriole were always at the same stage of development, it was inferred that morphogenesis at any one site was synchronous. These workers also observed occasional formation of transitory rudimentary cilia, often with abnormal and incomplete microtubule patterns in cross section. These appeared several days before the main ciliature formed. Dippell (1968) detailed the sequential assembly of basal body structure in Paramecium, as shown in Fig. 2. Basal body assembly in this ciliate is under very tight spaital and temporal control. New basal bodies form immediately anterior to an existing adult basal body but separated by a few hundred angstroms; the majority form in a 20-minute period, 50 minutes before the completion of cell division. Dippell did not find the diverse electron-dense aggregates reported in the vertebrate studies. The first structure she observed was the direct precursor of the basal body cylinder, a flat disk of dense, fibrous material with no discernible substructure. Microtubule assembly always started at a specific point and proceeded around the disk, forming a ring of nine singlet tubules. B-tubule assembly then started, often before completion of all the A tubules and not always sequentially. Dippell observed short fibers between adjacent tubules in the beginning stages, which later disappeared; she speculated that these fibers functioned in

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ELIZABETH C. RAFF

FIG. 2. Morphogenesis of basal bodies in Parumetlium. (a) Formation of the first tubule. 140.000. (b) A ring of singlet tubules partially completed. X 138,000. (c) Formation of the B tubules; doublets are partially completed. X 140,000. (d) Adult basal body (in cross section) with a new basal body anterior and at right angles. X140,ooO. (e) Four generations of basal bodies. X

~68,000. Reprinted from Dippell (1968), with permission.

development of the ninefold symmetry. After the triplet tubules formed, the other internal structures of the central cartwheel appeared. New basal bodies moved to the cell surface, where the cilia ultimately formed. Dippell often observed cytoplasmic microtubules associated with both growing and mature basal bodies; these were never continuous with basal body microtubules but inserted or originated in dense material around the basal body. The position of an existing basal body determined the position of the new one; Dippell occasionally ob-


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served new basal bodies forming near immature ones that had not yet moved toward the cell surface. A sequence of events similar to that reported by Dippell was observed by Johnson and Porter (1968) during formation of basal bodies at cell division in the unicellular biflagellate alga Chlamydomonas reinhardii. The earliest stage in basal body development they observed was a ring of nine singlet tubules. Cavalier-Smith (1974) reported that basal body morphogenesis in this organism, as in the vertebrate studies, could proceed through two alternative pathways. In vegetative cells flagella are resorbed prior to cell division, but the basal bodies remain attached to the plasma membrane.. A new basal body arises close to the old one, physically attached to the wall of the old one by amorphous material. In zygotes the basal bodies and associated structures as well as the flagella disappear, and there is no trace of the flagellar apparatus throughout zygospore maturation; basal bodies are then assembled de n o w close to the plasma membrane during zygospore germination. Basal body assembly is apparently the same in both the vegetative and sexual cell cycles, the first recognizable intermediate being the ring of nine singlet tubules around a central cartwheel structure. Cavalier-Smith also occasionally observed disklike structures which may have been earlier stages. The B and C tubules were apparently added on together, followed by elongation of the cylinder and finally the appearance of accessory structures such as roots and striated connections. As in other organisms, flagellar outgrowth began only after complete assembly of the basal body and its attachment to the plasma membrane. The cellular control over the number of flagella and accessory structures appears to be more stringent than control over the number of basal bodies; daughter cells may have four basal bodies but rarely more than one pair of flagella and associated structures. During mitosis, when the basal bodies are free of flagella, their identification as such or as centrioles is ambiguous. However, although they may sometimes be physically close to the mitotic apparatus, they do not function as mitotic centers (Cavalier-Smith, 1974; Coss, 1974; Johnson and Porter, 1968). Johnson and Porter (1968), however, suggested that their position may be directly involved in determination of the plane of cell division. Gould (1975) examined intermediate structures in preparations of basal bodies isolated from Chlamydomonas, as shown in Fig. 3. He also confirmed that the primary component of basal bodies is in fact the structural microtubule protein tubulin. Gould suggested that his results “revive” the possibility of the generation of new basal bodies by direct nucleation from an existing basal body. His electron micrographs showed that isolated basal body pairs have two probasal bodies attached to them through a complex of associated structures; just after cell division the probasal body consists of a ring or annulus of nine “dots” (rudimentary microtubules) connected to the mature basal body by fibers. This annulus

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FIG.3. Isolated basal bodies from C. reinhardii. Stages in morphogenesis. (a) Basal body pair isolated after cell division with two probasal bodies each consisting of an annulus of nine components, two of which are connected by long fibers to the proximal end of one of the mature basal . c) Basal body pairs isolated before cell division, showing elongation of the bodies. ~ 3 3 , 0 0 0 (band probasal bodies. ~ 3 3 , 0 0 0 (d) . Basal bodies isolated at the onset of mitosis, showing completion of probasal body maturation. X33.000. Reprinted from Gould (1975), with permission.

was not visible in thin sections of cells, either because it is too thin or possibly because it is unstable. Just before the next cell division the probasal body elongates and forms a new mature basal body; at this time Gould was able to distinguish that each dot is in fact a triplet microtubule, several of which are connected to the proximal end of the mature basal body by fibers. Gould interpreted his data to mean that development of the probasal bodies proceeds through simultaneous assembly of the A, B, and C tubules, but that the A tubules elongate some-


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what faster than the others so that the growing end of the probasal body might appear in cross section to represent a nine-singlet tubule structure. Basal body morphogenesis has been observed in a variety of other organisms; in many cases it appears to occur very rapidly without obvious intermediate forms. Millecchia and Rudzinska (1970) observed that basal body morphogenesis in the suctorian Tokophyryu infusionum proceeded similarly to that in Purumecium; new probasal bodies arose adjacent to mature ones, the A tubules being formed first. Allen (1969) reported that the earliest stage he observed in basal body morphogenesis in Tetruhymenu was a structure containing nine singlet tubules. Fulton and Dingle (1971) reported that no structure resembling a centriole or basal body could be found in the ameboid form of the ameba-flagellate Nuegleriu but that during transformation to the flagellated morphology basal bodies appeared about 10 minutes before the flagella themselves. These investigators found no structural precursors, although it appeared that basal bodies arose within the cytoplasm and then moved to the cell membrane. Outka and Kluss (1967) similarly did not observe basal body precursors in amebas of the ameba-flagellate Tetrumitus rostrutus, but during the ameba-to-flagellate transformation they found basal body-like structures in association with the nuclear membrane or with dense bodies in the cytoplasm. More recently, Ash and Stephens (1975) found that, during ciliagenesis in the gill of the bay scallop, Aequipecten irradiuns, the formation of basal bodies combined two aspects of morphogenesis seen separately in other studies. First, as in vertebrate studies, they observed the appearance of a complex of dense granules but thereafter, as in Naegleriu, mature basal bodies appeared very rapidly with no obvious organized intermediate stages. Basal body formation was not synchronous within a cell. As in other systems, the elaboration of accessory basal body structures and ciliagenesis was initiated after movement of the mature basal body to the cell surface. The above-mentioned studies, although differing in detail, give a fairly unified idea of the sequential assembly of basal bodies and centrioles. There are, however, several examples of different-I am tempted to say stranger-modes of formation of basal bodies, particularly in the development of multiple flagellated plant sperm (see reviews by Hepler and Palevitz, 1974; Paolillo, 1975). For example, Mizukami and Gall (1966), and more recently Hepler (1976) and Myles and Hepler (l977), have described spermiogenesis in the fern Marsilea. Marsilea sperm have over 100 flagella, the basal bodies of which arise from the blepharoplast, a spherical structure 0.8 pm in diameter formed before the last cell division in developing spermatids from a solid sphere of material of moderate electron density and complex substructure. This structure separates into two blepharoplasts which appear to serve as microtubule-organizing centers during assembly of the mitotic spindle, although they do not remain as the focal points of the mitotic tutubles after prophase. During metaphase the blepharoplast becomes hollow, and at this time the walls can be seen to consist of radially

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arranged, closely packed procentrioles. After cell division, the blepharoplast fragments and the procentrioles are released into the cytoplasm; they elongate and migrate to the cell surface, and the flagella are assembled. Similar events occur during spermiogenesis in the cycad Zamia, in which the sperm have upward of 20,000 flagella and the blepharoplast is huge, 10 p m in diameter (Mizukami and Gall, 1966), and in Equisetum (Duckett, 1973). B. THEASSEMBLYOF

THE

AXONEME OF CILIAA N D FLAGELLA

The assembly of the cilia or flagella axoneme may be thought of as a continuation of the process initiated by the formation or activation of the microtubuleorganizing center, in which the formation and placement in the membrane of the basal body are the next steps. The location and orientation of the axonemal shaft are specified by the location and orientation of the basal body; furthermore, although transitional structures are formed at the junction of the axoneme and the distal end of the basal body, the outer nine doublet tubules of the axoneme are specified by the A and B tubules of the basal body triplet tubules. The positioning of the basal body at the cell surface appears to be achieved by fitting either the basal body itself or some of the accessory structures into a specific site at or near the membrane [for example, Cavalier-Smith (1974) showed that in Chlamydomonas the basal body is attached to the cell membrane by the transitional fibers]. In some cases new basal bodies arise next to old ones, which places them close to the site they will occupy when mature; in others, the basal body arises in the cytoplasm and migrates to the cell surface. A mechanism may be envisioned in which the basal body travels along the cell surface until it “locks” into the specific site, as has been shown for another organelle, the trychocyst, in Paramecium (Pollack, 1974; Sonnebom, 1974). Thus control over the outgrowth of the axoneme is primarily temporal. The control over timing may be complex. For example, Nanney (1975) observed that in Tetrahymena new basal bodies were assembled adjacent to existing ones a complete cell cycle before they become ciliated. As discussed above, Sorokin (1968) and Anderson and Brenner (1971) observed that, when ciliagenesis took place too early, the resulting axonemes were transitory and defective. The regulation of ciliagenesis involves not only the initiation of axoneme assembly, but also determination of the final length of the axonemal shaft and in many cases disassembly of the axoneme as well, as many cells resorb their axonemes at some point in their life cycle (e.g., Chlarnydornonas before division). Electron microscope studies of basal body morphogenesis and subsequent ciliagenesis suggest that amorphous or fibrous material, similar to that identified as the precursor material for centriole formation, is the precursor material from which the axoneme is assembled (Ash and Stephens, 1975; Cavalier-Smith, 1974; Steinman, 1968). Outka and Kluss (1967) observed that during flagellar


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growth in Tetrumitus rostrutus a membrane-bounded vesicle which became filled with fibrillar material formed at the tip of the shaft; this vesicle disappeared as the flagella grew, suggesting incorporation of the material into the axoneme. The regeneration of amputated cilia and flagella has provided an ideal model system. Initial studies by Rosenbaum and Child (1967) and Rosenbaum and Carlson (1969) showed that, after amputation of the flagella of Ochromonas, Euglenu, and Astusiu or the cilia of Tetruhymena, there was a lag during which protein synthesis appeared to be required; thereafter, elongation proceeded at a rate which constantly decelerated as the final length was approached. Rosenbaum et ul. (1969) and Coyne and Rosenbaum (1970) extended the observations to Chlumydomonus. They reported that, in a single cell, assembly of one flagellum could occur at the same time as disassembly of the second. If only one of the two flagella were removed, the remaining one shortened at the same time as the amputated one began to elongate. When protein synthesis was inhibited with cycloheximide, the regenerating flagella reached only about one-third of the normal length; in cells with only one amputated flagellum, the length of the two flagella finally regenerated in the presence of cycloheximide depended on how long the stump of the remaining one had been. Tamm (1967) also studied control of flagellum length. He found that the initial rate of elongation of regenerating flagella of Puranema depended on the length of the stump of the amputated flagellum; that is, the longer the stump of a partially amputated flagellum, the slower the rate of regeneration. The final flagellum length was constant. Thus the cell appeared to “know” how long the flagellum was. Another interesting example was reported by Kerr (1972), who studied elongation of the flagella of the slime mold Didymium nigripes. This organism has two flagella of unequal length; these not only elongated at different rates but reached the maximum rate of elongation at different times. Cycloheximide caused flagellar growth to cease. She concluded from the complexity of the growth kinetics that the mechanism of regulation of elongation did not simply depend on the diffusion of subunits to the growing end of the axoneme nor was it a simple function of the total length. Bums (1973) observed that the regeneration of cilia in gastrulae of the sea urchin Tripneustes grutillu also took place after a lag and proceeded at a decreasing rate so that the final length was reached asymptotically. Interestingly, “animalized” cilia, which were up to three times the length of normal cilia, elongated at the same initial rate but had an extended period of elongation. Thompson et ul. (1974) observed the regeneration of Tetrahymena cilia in a scanning electron microscope study. They reported that ciliagenesis proceeded synchronously in recently divided cells but asynchronously in others and that, although oral and somatic cilia elongated at the same time, the rate of elongation of oral cilia was faster. Rannestad (1974) also investigated the regeneration of

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cilia in Tetrahymena. He found that, similar to the situation in Chlamydomonas, if only 75% of the cilia were removed, the remainder were resorbed before regeneration of the whole ciliature; again, however, some cilia began to regenerate even before all the remaining old cilia were completely resorbed. Regeneration required protein synthesis or resorption of old cilia; partially but not completely deciliated cells regenerated cilia in the presence of cycloheximide. However, the volume of cilia regenerated (about 90% of the cilia) in the presence of cycloheximide was greater than the volume resorbed (about 25%). Rannestad concluded that material contributed by the resorbed cilia potentiated the assembly of cilia from pools of already existing precursors. Certainly his results and those of Rosenbaum and co-workers indicate both the need for synthesis of some but not all axoneme components and the possibility of reutilization of resorbed components. All these studies illustrate the complexity of control and the possibility of separate regulation of similar organelles in a single cell. An obvious possibility for part of the control of assembly of the axoneme is the synthesis of a certain cornponenet in limiting amounts. This may occur during ciliagenesis in the sea urchin embryo. Regulation is not, however, achieved through the modulation of levels of tubulin. In an early study Auclair and Siege1 (1966) showed that even repeated regeneration of the cilia of gastrula stage embryos could take place in the presence of actinomycin D and puromycin (which decreased total protein synthesis by 90%), thus suggesting that cilia were assembled from a preexisting pool of subunits. Work in several laboratories has shown that a tubulin pool is made during oogenesis and is subsequently maintained throughout early development (Bibring and Baxandall, 1977; Borisy and Taylor, 1967b; Cognetti et a/., 1977; Raff, 1975; Raff and Kaumeyer, 1973; Raff et al., 1971, 1972, 1975). Work by Stephens (1972b, 1977b) has shown that, while pools of tubulin and several other axoneme components are maintained and synthesized at constant rates, the initiation of ciliagenesis is accompanied both originally and during regeneration by the synthesis of limiting amounts of two minor components. Several studies have examined the synthesis of tubulin (but not of other axoneme proteins) during the growth of cilia or flagella in other organisms. Nelson (1975) showed that, as in the sea urchin, ciliagenesis in Tetrahymena utilized preexisting stores of tubulin. In some cases, however, the onset of ciliagenesis is marked by tubulin synthesis. Dirksen and Staprans (1975) found that, although there were tubulin pools present prior to ciliagenesis in the mouse oviduct, 90% of the tubulin in 3-day-old and 75% in 5-day-old mouse oviducts was newly synthesized. Weeks and Collis (1976) found that tubulin synthesis was induced when flagella were removed from Chlamydomonas cells. However, Weeks et al. (1977) observed that the induction of tubulin was not dependent on the actual utilization of tubulin stores during flagella regeneration, both because


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of the timing of the induction-it took place before the tubulin pools were depleted-and because inhibition of regeneration with colchicine did not inhibit the induction. These investigators suggested that a feedback-type mechanism might operate once tubulin levels reached a certain point since, when flagella regeneration was blocked with colchicine, the induced synthesis decreased earlier than in the controls. Finally, Fulton and co-workers found that tubulin utilized in the formation of flagella was specifically synthesized during transformation from the ameboid to the flagellate form in Naegleria gruberi (Fulton and Kowit, 1975; Fulton and Simpson, 1976; Kowit and Fulton, 1974a,b). It is clear that overall assembly of the flagella or cilia shaft proceeds from the basal body outward. Oddly enough, however, it is still unclear whether microtubule assembly takes place distally (by the addition of subunits at the growing tip), as has been implicitly assumed in many discussions, or proximally (by the insertion of subunits at the basal body end). Early studies of flagellar growth in Ochromonas and Chlamydomonus by light microscope autoradiography indicated that, while most of the radioactivity from radioactively labeled amino acids incorporated into regenerating flagella appeared in the distal half of the organelles, some label always appeared in the proximal half of the shaft (Rosenbaum and Child, 1967; Rosenbaum et al., 1969). The possibility existed that some or all of the label seen in the proximal part of the flagella was due to proximal growth of the membrane or other nonmicrotubule parts of the axoneme. Witman (1975) repeated these experiments, pulse-labeling regenerating flagella of Chlamydomonas; he then isolated the flagella and purified the outer nine doublet fibers and examined the incorporation by electron microscope autoradiography . He found that 65% of the grains appeared over the distal half of the flagella and 35% over the proximal half; he concluded that the outer doublet tubules are primarily assembled at the tip, but the results were still ambiguous. More recently, Dentler and Rosenbaum (1977) reexamined flagellar growth and shortening in Chlamydomonas by electron microscopy and concluded that, while the outer doublet tubules appeared to be assembled by distal addition of subunits, the central pair of singlet microtubules appeared to be assembled by proximal insertion of subunits. They reached this conclusion partly through in vitro experiments. A central “cap” attaches the distal end of the central microtubules to the tip of the flagellar membrane; this structure stays in place throughout both elongation and resorption of the flagellum. There are also distal filaments present on the ends of the A tubules of the outer doublets. When isolated axonemes were attached to grids and incubated with purified brain tubulin, assembly occurred at the distal ends of the A tubules (in spite of the distal filaments), but the cap on the central pair prevented assembly. However, if the cap was lost during the isolation procedure, assembly of tubulin subunits took place at the distal ends of the central pair. Dentler and Rosenbaum pointed out that assembly of the central microtubules might take place distally in vivo even in the presence of the cap, but

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that attachment to the grids might in some way prevent the process in vitro. Nevertheless, their results reopen the old question concerning the mechanism of assembly of cilia and flagella in vivo. Control of the disassembly of flagella is a problem that has been treated as the obverse of assembly, although the regulatory mechanisms may in fact be different. In most of the studies cited above, flagellar regression, like growth, appeared to take place gradually and sequentially at the tip, in the reverse order of assembly (see, for example, Cavalier-Smith, 1974). A variation on this was observed by light and electron microscopy by Ishigami (1977) in the myxomycete Sternoniris pallida during transformation from the flagellate to the ameboid form. Within 60 seconds the entire axoneme was resorbed into the cell body by fusion of the flagellar and cell membranes. At first the entire axoneme was visible in the cytoplasm (sometimes still beating); then it disintegrated over about 90 minutes-B tubules first, and then A tubules, spokes, and central tubules in that order. Cellular control over the disassembly process was indicated by the fact that cytoplasmic microtubules and the basal bodies apparently persisted much longer. This organism has two flagella, a long one which beats and a short one which does not; the short one is always retracted first.

c. THEMORPHOGENESIS OF MICROTUBULE-CONTAINING ORGANELLES IN PROTOZOA

In addition to simple cilia and flagella, many specialized protozoan organelles are constructed from specialized cilia or other patterned arrays of microtubules. The oral apparatus of Tetruhyrnena consists of closely arrayed cilia forming four “membranes” which are the basis of the organism’s generic name, plus additional microtubules. These specialized cilia are under different control than the regular somatic ciliature. Williams and Frankel (1973), Williams and Nelson (1973), and Williams (1975) have described the assembly of this organelle. A new oral apparatus is formed at cell division and also under conditions of amino acid starvation, when it is resorbed and then redifferentiated or replaced. At cell division, a new oral apparatus, which will become the oral apparatus in the posterior daughter cell, forms behind the future site of cell cleavage; in oral replacement the new apparatus forms just anterior to the site of the old one, which is resorbed. During resorption of the old apparatus the cilia are apparently withdrawn into the cytoplasm and disassembled; meanwhile basal bodies proliferate rapidly, new basal bodies being formed adjacent to mature ones. Very little protein synthesis is necessary for oral replacement; these investigators speculate that basal body proliferation may be controlled by the synthesis of a small number of regulatory proteins. The complexity of microtubule regulation is vividly obvious in this organism. During the final quarter of the cell cycle, a new oral apparatus begins to form in the posterior of the cell. The old apparatus


17

meanwhile migrates to the cell surface (i.e.. the “mouth” flattens out) and undergoes regressive changes; the undulating membrane cilia are lost, and the buccal membranelle cilia shorten. Thus for a time two identical microtubulecontaining structures are present in a single cell, one in the process of assembly and the other in the process of disassembly. Ultimately the regressing old apparatus and the forming new apparatus reach the same stage, and then during cell cleavage both develop synchronously to the final completely differentiated structure. Tucker (1970, 1971) described the morphogenesis of the cytopharyngeal basket, the mouth structure of another ciliate, Nussula. This organelle is constructed of a cylinder of rods (the basket units) each of which is composed of a bundle of microtubules aligned in parallel along their long axes. Similarly to the situation in the oral apparatus of Tetruhymena, disassembly of the old basket occurs simultaneously with assembly of two new ones. During interphase the cytopharyngeal basket is located immediately anterior to a pore. Before cell division the old basket detaches and “floats” into the cytoplasm, where it breaks down. Meanwhile, at the anterior of the dividing cell a new basket forms at the site of the old basket, and a new pore appears just behind it; at the posterior, a second new basket forms just in front of the old pore. Thus in the two resulting daughter cells, the relative arrangement of basket and pore is maintained, their positions being determined by membrane sites. Morphogenesis of the basket is extremely complicated. At the proximal end of the basal bodies (which bear cilia at their distal ends) a structure called the laminated cap appears. It is an electron-dense, layered plate from which microtubules in a highly ordered parallel array emerge; Tucker has observed that the microtubules appear to arise from the middle layer of the cap and pass through the bottom layer. Each array ultimately forms one basket unit, as shown in Fig. 4. The units detach from the basal bodies but retain the cap structure; they then align in a row which curls in from both ends, forming the cylindrical basket. During the rolling up of the basket a reticulum of microtubules forms just beneath the pellicle in the region of the basket. Most of these tubules are caught inside the basket and resorbed; they possibly represent a “scaffold” around which the basket is formed. Occasionally Tucker observed mistakes; sometimes one of the units moved too fast or too slow and was “caught” in the middle of the forming basket or else left behind in the cytoplasm. The units left behind persisted in the cytoplasm near the basket until the next cell division, when they were disassembled along with the old basket. The arrangement of the units in mature baskets does not reflect the arrangement of the basal bodies near which the laminated cap and associated microtubules arose. The basal bodies themselves remain in place, the cilia they bear becoming the paroral ciliary row. The control over ciliagenesis also is complex in this organism; different classes of cilia persist andor are generated at different times in the cell cycle. Tucker pointed out that a mechanism of microtubule-nucleating

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ELIZABETH C. RAFF

FIG.4. Morphogenesis of the cytopharyngeal basket of Nussula. Longitudinal section through the top of one basket unit showing the microtubules, laminated cap, basal body, and cilium. x I13,OOO. Reprinted from Tucker (1970). with permission.

C O N T R O L OF MICROTUBULE ASSEMBLY

19

sites plus diffusible subunits could not generate the events he observed. Rather, this complex control must also involve localization of the structural components or of the signaling or regulatory factors, or both. This interesting system has been further described more recently by Tucker et al. (1975) and Pearson and Tucker (1977), who observed in detail the bundles of microtubules arising from the laminated cap. The tubules in the units are packed in a hexagonal pattern; the pattern is disrupted if the organelle develops in the presence of colchicine. By comparing the development in the absence and in the presence of colchicine, they concluded that the pattern is determined by the specification of the site of origin of each tubule; that is, the cap comprises a microtubule-nucleating template. The microtubules in the bundle are subsequently connected by cross-linking structures which maintain the hexagonal cross-sectional pattern permanently. However, it is the nucleating sites in the laminated cap and not the links which establish the pattern, and the pattern is at first simply maintained by the close packing of the growing microtubules. Thus the dense sheets which arise at the proximal end of the basal bodies are microtubule-organizing centers in the classic sense. There are no data as yet on the biochemical nature of this material or how it is regulated. The function of the adjacent basal bodies remains obscure; it seems possible that they may serve as a means for localizing formation of the microtubule bundles rather than participating in generating them. Tucker (1977) reviewed the general problem of how the shape and pattern of microtubule arrays can be specified and concluded that, while the patterns themselves may be specified in several ways (e.g., self-linking of tubules, simple close-packing arrays, or template-type nucleation), additional regulatory mechanisms including compartmentalization of components must also exist. A third interesting protozoan model system is the formation and distribution of microtubules in Ochromonus, described in an elegant study by Bouck and Brown (1973a,b; Brown and Bouck, 1974). This organism is a biflagellate which maintains a teardrop shape although it lacks either a pellicle or cell wall; at the anterior of the cell is a “beak” from which the flagella emerge, and at the posterior is a narrow extension of the cytoplasm, the “tail.” Bouck and Brown have observed that the shape of the cell is maintained by two sets of cytoplasmic microtubules which appear to be under separate cellular control even though the microtubules in the two sets are physically very close to each other. The axoneme microtubules are the only microtubules directly connected to the two basal bodies, but both sets of cytoplasmic microtubules are associated with several fibrous structures which are themselves associated with the basal bodies. There are two striated fibers; one connects the two basal bodies and the second, the rhizoplast, shown in Fig. 5, extends from the region of the basal bodies toward the nuclear membrane. The rhizoplast consists of amorphous electron-dense material arranged in cross-

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ELIZABETH (3. RAFF

FIG. 5 . Rhizoplast microtubules in Ochrornonas. (a, During interphase. The rhizoplast extends over the nuclear surface and is associated with a Golgi complex on the upper surface, while the microtubules extend from the lower surface. X 56,000. (h) During mitosis. The basal body, to which the mitotic rhizoplast remains attached, can be seen in the upper left. x20.800. Reprinted from Bouck and Brown (1973a). with permission.


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ELIZABETH C. RAFF

bands; the upper surface is associated with the Golgi complex, and from the lower surface extends the set of microtubules which determine the shape of the tail. There are also several unstriated fibers associated with the basal bodies. One set of these, the kinetobeak fibers, consists of amorphous material plus arrays of microtubules which begin in parallel and then curve and splay outward; these microtubules determine the beak shape. The independent regulation of these two sets of microtubules was determined from the fact that they were differentially sensitive to pressure and colchicine and that both disassembly and assembly of the two sets occurred separately in a specific sequence. In cells exposed to colchicine or put under increased pressure, the cytoplasmic microtubules disassembled with a concomitant loss of the typical cell shape, the process proceeding from posterior to anterior. When colchicine was removed or the pressure released, the microtubules reassembled and the cell shape was restored. Reassembly of the kinetobeak tubules occurred first, within 10 minutes, and after 15-30 minutes the rhizoplast microtubules reassembled. The rhizoplast itself was not altered by microtubule-depolymerizing treatments nor were the relative locations of the rhizoplast and the kinetobeak nucleation regions. A similar sequence of disassembly occurs before cell division. During mitosis the spindle microtubules are attached to a structure identical to the rhizoplast. After division the two sets of microtubules reappear, forming the beak first and then the tail. Bouck and Brown concluded that in Ochrornonas the two nucleation sites control the timing, orientation, and pattern, as well as position, of the microtubule arrays. Finally, the axopodia of heliozoan protozoa have provided a model system for study of the regulation of labile microtubules. These organelles are rods resembling miniature sea urchin spines. Tilney and co-workers showed that microtubules were responsible for both the production and maintenance of the form of the axopodia of Echitiosphaerium (formerly called Actirzosphaerium), which consist of microtubules packed in parallel longitudinally with a cross-sectional pattern of an interlocking double spiral (Tilney, 1968a,b, 1971a; Tilney and Byers, 1969; Tilney and Porter, 1965, 1967; Tilney et al., 1966). While the axopodia are permanent features of the protists that bear them, they are dynamic organelles in that the cell constantly changes their length during feeding or as a consequence of changes in the environment, by assembly and disassembly of the component microtubules. Experimentally they could be induced to disassemble by the application of cold, pressure, or colchicine, or to reassemble after release from these treatments, and were stabilized by deuterium oxide, as is typical of labile microtubule systems. Tilney and co-workers found that assembly and disassembly of the microtubules takes place sequentially at the tip of the organelle and that the subunits are reused during reassembly of the organelles. They concluded that in this organism the cross-sectional pattern is determined by specific cross-bridging of the growing microtubules.


23

A mechanism explaining how the cross-sectional microtubule pattern in

Echinosphaeriurn axopodia is generated by self-linkage was proposed by Roth

and co-workers; they formulated the “gradion” hypothesis, according to which the binding of the first linker to a particular tubulin subunit allows the binding of the second, and so on, in an allosterically induced conformational gradient (Roth and Pihlaja, 1977; Roth et al., 1970). Ockleford and Tucker (1973) observed contraction and regrowth of the axopodia of a related organism, Actinophrys sol. They reported that as in the assembly of flagella, the rate of elongation of the axopodia decreased as the final length was approached. Regrowth of a single axopodium contracted during feeding proceeded at the same rate as regrowth of the entire set of axopodia after cold treatment. Echinosphaeriurn and Acrinophrys belong to the Actinophryida, heliozoa in which the axopodia arise independently either at the nuclear membrane or in the cytoplasm. In the Centrohelida, the axopodia arise from the centroplast, a dense structure in the center of the cell, and the microtubules composing them are in hexagonal or triangular cross-sectional patterns (see Bardele, 1977, for a brief review of the heliozoan groups). Tihey (1971b) studied reformation of the axopodia of Ruphidiophrys after disassembly of the microtubules by cold treatment; the microtubules indeed initiated from the centroplast (which in this organism is highly electron-dense and without discernible substructure), but pattern formation did not begin until the microtubules had extended some distance from the centroplast. The pattern, as in Echinosphaeriurn, was generated by specific bridging of the microtubules; each microtubule could be bridged to four others. Tilney concluded that, once one bridge was attached, the position of the others was determined, similar to the mechanism proposed by Roth et al. (1970). Bardele (1977) concluded that, while in some Centrohelidan heliozoa the axopodial pattern was specified by self-linkage, as observed by Roth and Tilney, in others it appeared that a template-driven nucleation followed by bridging occurred, similar to the case in Nussula. Bardele’s electron micrographs of the centroplast of species of Heterophrys show that it is a “hollow” sphere containing an electron-dense striated disk(s) in the center; the axopodia microtubules radiate from the electron-dense surface of the sphere, but their mode of attachment is unclear because their proximal bases are surrounded by amorphous electron-dense material. A N D DISASSEMBLY O F LABILE MICROTUBULE ARRAYS: D. THEASSEMBLY THEMITOTICAPPARATUS

The literature on mitosis is at least as extensive as the literature on microtubules, part of which it includes. The classic review by Mazia (1961) gives a

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ELIZABETH C. KAFF

comprehensive historical view of observations of mitosis. Consideration of the mitotic apparatus has focused on two problems. One is the mechanism by which chromosome separation is achieved. The two basic theories of chromosome movement are, first, the dynamic equilibrium theory proposed by Inoue (1964) and Inoue and Sat0 (1967), whose elegant studies using birefringence microscopy established the labile nature of the mitotic fibers and who suggested that chromosome movement results directly from equilibrium assembly and disassembly of microtubules from a pool of subunits, and second, the sliding-filament model proposed by McIntosh et al. (19691, who suggested that movement occurs as a result of microtubules sliding relative to each other, similar to the mechanism accepted for the bending of cilia and flagella. As yet, however, no single hypothesis as to how microtubules function in chromosome movement explains all the data (for recent summaries and discussions of this problem, see Bajer and MolC-Bajer, 1975; Inoue, 1976; Inoue and Ritter, 1975; McIntosh et al.. 1975b, 1976; Nicklas, 1975; Salmon, 1975c, 1976). Recent evidence has suggested the possibility that actin may alsci be involved in the production of chromosome movement (Cande et al., 1977; Forer, 1976; Sanger and Sanger, 1976). The second problem of course is how the mitotic apparatus is formed and how the cell regulates its appearance, an important solo part in the overall orchestration of cell division. It may well be that how the mitotic appararatus functions and how it is regulated are not independent questions but, since the data are not complete, it seems useful and possible to consider the latter problem separately. Formation of the mitotic apparatus depends on the formation and/or functioning of microtubule-organizing centers. In a typical mitotic apparatus microtubules arise at two different kinds of sites, at the mitotic poles and at the kinetochores of the chromosomes. The formation, morphology, and mode of function of various astral and anastral mitotic spindles were reviewed by Bajer and Mole-Bajer (1971) and Nicklas (1971). As these workers pointed out, the point of origin of polar mitotic tubules may he unclear, since a variety of structures (or no structure) exists at mitotic poles in different organisms, but kinetochore microtubules clearly arise at this differentiated area of the chromosome. Recent electron micrographs by Roos (1977) show the kinetochore of mammalian chromosomes in cross section to consist of outer and inner electrondense layers separated by a less dense middle region; it is biochemically as well as structurally differentiated, since cytological staining shows no chromatin at least in the outer and middle layers. The kinetochore microtubules arise directly from the dense material of the outer layer and do not extend into the other layers. This structure is reminiscent of other microtubule-organizing centers such as the rhizoplast of Ochromonas and mitotic centers in certain other organisms discussed below.


25

Since a centriole appears at each pole of an astral mitotic figure, it was assumed for a long time that the centriole was the actual focal point for the polar mitotic tubules and in some way produced them or caused their appearance. It is now abundantly clear that this is not so. For one thing, this proposal left unexplained anastral acentriolar mitotic figures, of which there are innumerable examples and which function perfectly well. Pickett-Heaps (1969, 1971, 1975a) has suggested that, while this structure in its role as a basal body is an example of a highly structured microtubule-organizing center, its association with the mitotic apparatus is in fact a mechanism to ensure its correct partitioning to the daughter cells during cell division. However, this rationalization is not entirely satisfactory either, since it is clearly possible for many cells to elaborate centrioles de n o w , and since even in a single organism centrioles may be present in some but not other mitotic figures. The centriole retains its position as the Mona Lisa smile of mitosis. Szollosi et crl. (1972) reported that, while centrioles are present at spindle poles of oogonia and oocytes of mice and other mammals, they are absent in meiotic figures subsequent to the pachytene stage. Their electron micrographs showed that at the time of germinal vesicle breakdown in mouse oocytes, aggregates of electron-dense fibrous material appeared, from which microtubules radiated forming acentriolar asterlike arrays; groups of these subsequently served as spindle poles. Recent studies by Berns and co-workers of centrioles and mitosis in rat kangaroo cells in culture have demonstrated that pericentriolar material but not the centriole is required for both assembly and function of the mitotic apparatus. The mitotic pole in these cells is of the typical astral type, consisting of a centriole surrounded by amorphous material from which the polar tubules extend. At prophase Berns et a / . (1977) irradiated with an argon laser microbeam one of the centriolar areas of cells sensitized to radiation by acridine orange treatment; the primary damage was dispersal of the pericentriolar material, possibly suggesting a nucleic acid component. These cells underwent nuclear breakdown, chromosome condensation, formation of the metaphase plate, and cytokinesis, but the chromosomes did not separate nor did any anaphase movements occur. Electron micrographs showed kinetochore microtubules on both sides of the chromosome mass, but the polar microtubules were not formed normally and microtubules were absent from the cleavage constriction. In a second series of laser microbeam experiments, Berns and Richardson (1977) directly irradiated the centriolar region during early prophase and observed mitotis in cells in which the centrioles were physically removed from the spindle, severely structurally damaged, or destroyed. The mitotic tubules remained focused on the pericentriolar material, and mitosis proceeded normally. This group also observed the dispensability of the centriole in tetraploid cells which underwent spontaneous reduction divisions;

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ELIZABETH C. RAFF

electron micrographs showed centrioles at only two of the four spindle poles formed, but the microtubules of all poles tenninated in pericentriolar-like material (Brenner et al., 1977). Newcomb (1969), Hepler and Palevitz (1974), and Pickett-Heaps (1975b) have reviewed the situation in plants; centrioles or basal bodies appear in algae and other lower forms, in ginkgos and cycads, but not in other gymnosperms and not at all in angiosperms. Higher plants have anastral mitotic figures, often with no discernible polar organizing site. In lower plants and other lower eukaryotes, a variety of specialized microtubule-organizing centers and mitotic spindle morphologies and modes of function occurs. Many of these organizing centers resemble some of those already mentioned and consist of layers or masses of electron-dense material from which microtubules initiate. In a review concerned with evolution of the mitotic apparatus, Kubai (1975) gives an exhaustive tabulation of spindle morphologies. Within the scope of this article this topic cannot be completely covered, but some particularly striking examples can perhaps serve to illustrate the point. Manton et a / . (1969) followed the development of the unusual mitotic spindle in spermatogonia of the marine diatom Lirhadesmiurn undulurum. No centriole was involved. Near the nucleus of nondividing cells was a spindle precursor consisting of a rectangular body composed of a series of parallel, electron-dense plates or layers. At prophase a spindle appeared between the precursor and nuclear envelope; microtubules arose out of extensions of the end plates of the precursor, which served as mitotic poles. During enlargement of the spindle the rest of the precursor structure disappeared, implying incorporation of precursor material into the developing spindle microtubules. Mitosis proceeded after the nuclear envelope disappeared, and the mitotic array “sank down” into the chromosomes. Zickler (1970) described the microtubule-organizing centers in four species of ascomycete fungi; again, centrioles were not involved. Mitosis in these cells occurred with the nuclear envelope intact. The microtubules of the mitotic or meiotic spindle assembled within the nucleus, arising at the poles from centrosomal plaques, sandwichlike structures with portions on both sides of the nuclear membrane. In some species, this structure is L-shaped, and part of it is not attached to the nuclear membrane but extends into the cytoplasm. Sometimes microtubules extend from the extranuclear side of the structure, giving the appearance of astral fibers. Similar spindle plaques consisting of layered, electron-dense bodies lying on the nuclear envelope function as spindle poles in the intranuclear mitotic apparatus of the yeast Succhuromyces cerevisiae (Moens and Rapport, 1971). After cell division, in each daughter cell a second spindle plaque arises adjacent to the old one; they then move apart and become the poles of the mitotic apparatus at the next division. Other organisms with layered, electron-dense microtubule-


27

organizing structures are the mushroom Boletus rubinellus (McLaughlin, 1971), the slime mold Physarum polycephalum (Tanaka, 1973), and the cowpea rust fungus, Uromyces phaseoli (Heath and Heath, 1976). Spherical microtubule-organizing structures resembling the blepharoplast of Marsilea have been observed in marine protozoa. Perkins (1970) described mitosis in a species of Lahrynthub; in vegetative cells the spindle microtubules directly attached to and presumably arose from two electron-dense spherical masses 200-300 nm in diameter. These aggregates had a central cartwheel structure similar to that in centrioles but lacked the nine triplet tubules. Tippit and Pickett-Heaps ( I 977) described a large, extremely complex spherical microtubule center in the pennate diatom Surivella ovulis; this organelle was present during interphase as a dense mass uniformly granular in substructure, from which extended a few microtubules. In early prophase a spindle formed beneath it, the microtubules of which extended between two dense end plates. As the spindle elongated, the spherical microtubule center dwindled and disappeared; a new one later formed de novo in each daughter cell. Other large, atypical microtubule-organizing structures have been observed in flagellates inhabiting the gut of wood roaches and termites. Grimstone and Gibbons (1966) described the ultrastructure of the centriolar apparatuses in Trychonympha and Psuedotrychonympha, which consist of long fibrillar rods lacking any centriolelike substructure but which serve as mitotic centers during cell division. Tamm and Tamm (1973a,b) described a similar apparatus in Deltotrychonympha and Koroga. Interphase cells contain two club-shaped bodies of fibrillar or granular material, with no discernible substructure or association with microtubules, connected to each other and arranged at right angles, reminiscent of a typical centriole arrangement; at division the two structures separate and serve as mitotic poles, spindle microtubules being assembled from the ends and radiating toward the nucleus. These organisms have the remarkable feature of containing hundreds of thousands of basal bodies of unknown function free in the cytoplasm, 500,000 to 700,000 immature basal bodies being arranged in chains and about a fifth as many mature organelles scattered singly (Tamm, 1972). Unstructured sites similar to the pericentriolar material have also been reported. Pickett-Heaps and Fowke (1970) described the events of microtubule assembly in the alga Closteriutn littorale, in which the mitotic spindle components apparently are altered after cell division and reutilized to reestablish cell organization. During mitosis, microtubules emerged from the mitotic centers, which consist of regions of granular material. At telophase, the spindle microtubules disassembled, but the mitotic centers persisted and in each daughter cell moved to the side and migrated along the chloroplast. Microtubules then extended in parallel from the organizing center toward the nucleus, forming a cylinder through which the nucleus moved, repositioning itself in its original place in the cytoplasm.

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A series of experiments indicates that in some organisms the two classes of mitotic microtubules have somewhat different properties and thus may be subject to differential control. That is, kinetochore microtubules appear considerably more stable than polar microtubules. Brinkley et al. (1967) found that, if Chinese hamster cells in culture were treated with low levels of colcemid, monopolar spindles formed in which polar microtubules were lacking but the chromosomes were attached by kinetochore microtubules radially around the pair of unseparated centrioles. Apparently the position of this aberrant mitotic apparatus was determined by the position of the centrioles. When the colcemid was removed, a normal spindle formed as interpolar microtubules formed and the two poles separated. Brinkley and Cartwright (1975) observed that in cultured rat kangaroo fibroblasts not only were the kinetochore tubules more stable than the polar tubules but that the lability of the polar tubules changed during the course of mitosis. Salmon (1975a,b,c) and Salmon et a / . (1976) observed that kinetochore tubules of several species were less sensitive to depolymerization by increased pressure than were polar tubules; they speculated that the greater stability may simply reflect the number of "attached" ends (kinetochore tubules extend to the poles and do not have "free" ends in the cytoplasm as do polar tubules). Essentially all observations on the assembly of the mitotic apparatus are consistent with what has become dogma, that is, that the mitotic apparatus is assembled out of a preexisting pool of tubulin subunits and, as originally proposed by Inoue (1976), the tubulin subunits are in equilibrium with the assembled microtubules. Thus the control of its appearance involves mobilization rather than synthesis at least of the primary molecular components. Some experiments by Stephens (1972a, 1973) dealt directly with this question; he showed that not only the size but also the morphology of the mitotic apparatus of sea urchin embryos depend on the temperature at which they are grown. Eggs of the cold-water sea urchin Stronylocetitrotus droebachietzsis develop to normal plutei even at 0°C. Stephens found that in embryos grown at 8°C the mitotic spindle was of normal morphology with large polar asters, but that in embryos grown at 0°C the mitotic spindle was of the anastral type and in addition was much smaller. He maintained fertilized eggs at 0" or 8°C and then raised the temperature at division; in both sets of eggs the spindle size and birefringence increased. In 8°C eggs large asters formed, but only a few astral fibers formed in 0°C eggs. Both sets showed maximum spindle birefringence at 12"C, the upper temperature limit for viability of this species. The differences in mitotic apparatus size indicated the portion of the total tubulin available for utilization rather than the total size of the pool, since after slow perfusion with deuterium oxide the resultant increased spindle size was the same for eggs at both temperatures. McIntosh et a / . (1975a) studied the mitotic spindle in mammalian cells using rat kangaroo cells in culture. Their electron micrographs showed that in these cells the microtubules at the mitotic poles end (or, presumably, originate) in


29

masses of amorphous electron-dense material; there were no asters such as observed in marine eggs. The metaphase spindle formed over a period of about $5 hour. Since treating the cells with puromycin an hour before the start had no effect on its formation, they concluded that there were probably no translational controls on microtubule assembly; however, the level of inhibition of protein synthesis attained (80%) did not preclude the synthesis of some components. They noted that determination of the spatial location of the mitotic apparatus persisted through premature disassembly of the mitotic microtubules by cold, colchicine, or increased pressure; when the cells were released from these treatments, the mitotic apparatus reformed in the same orientation it had had previously. This observation is similar to that reported earlier by Goode (1973) in a study of the kinetics of reformation after cold-induced disassembly of the mitotic apparatus in the ameba Chuos carolinensis; he concluded that this process involved only the final steps in mitotic apparatus formation, that is, microtubule elongation rather than initiation. Goode also concluded that the rate of reassembly was consistent with the diffusion of subunits to a single growing point per microtubule. How the mitotic apparatus is positioned in the correct orientation is an important question about which unfortunately little is known. The position of the mitotic apparatus is crucial to proper determination of the daughter cells. From observations of mitosis and the subsequent planes of cell cleavage it has been inferred that in many cells the orientation of the mitotic apparatus determines the plane of cleavage. This has been directly demonstrated in eggs of marine invertebrates, in which physically shifting the position of the mitotic apparatus has been shown to cause an accompanying shift in the subsequent position of cleavage (see the review by Rappaport, 1971). The classic example illustrative of this point is the embryonic development of the snail Lirnnaea peregru (Morgan, 1927). The symmetry of the adult snail, including the direction of coiling of the shell, is determined by the direction of spiral cleavage, which is in turn determined by the orientation of the mitotic apparatus at the second cleavage. This symmetry determination is controlled by a pair of alleles of a single gene; the majority of individuals are coiled dextrally, the allele for which is dominant over the allele for sinistral coiling. This gene is a maternal effect gene; that is, the genotype of the mother entirely controls the direction of coiling of the offspring. Recently Freeman (1977) has found that injection of cytoplasm from eggs or early embryos of dextral individuals into eggs produced by females homozygous for the recessive sinistral coiling pattern causes them to develop with dextral coiling. The reciprocal injection (from sinistral into dextral eggs) had no effect. These results imply the action of a gene product present in the dextral but not the sinistral form. A mechanism in which orientation is not determined until after formation of the mitotic apparatus has been described in the onion Allium by Palevitz and Hepler (1974a,b). During guard cell division the cell plate forms along the

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ELIZABETH C. RAFF

longitudinal axis of the cells (whereas in other cell types division is transverse). In these cells, however, the position taken by the mitotic apparatus is apparently simply that in which it has the most “room” to function-obliquely or longitudinally across the cell. Mitosis proceeds until late anaphase or early telophase, at which point the entire spindle with the daughter chromosome sets reorients, the poles going to specified points on the cell periphery. Bands of microtubules appear at these points on the membrane, but how they participate in the reorientation is not clear. The reorienting movement is slow, requiring 15-20 minutes, but is specific; these investigators did not observe any cases in which the final position was “overshot. The movement was reversibly blocked by metabolic poisons and prevented by treatment with colchicine and vinblastine, in which case aberrant and misplaced cell plates formed. Several workers have concluded that in viva formation of microtubules involves the closure of sheets of filaments at the tips (or opening of the tubule during disassembly). Such intermediates would be seen in cross section as C-shaped. C-shaped microtubules have indeed been observed in sections of both isolated spindles (Cohen and Gottlieb, 1971) and mitotic cells (Jensen and Bajer, 1973); such profiles are most common near the equatorial region in metaphase, which presumably represents a region of active microtubule assembly andor disassembly. C-shaped cross sections interpreted as intermediates have also been observed during the rapid assembly of microtubules in mammalian blood platelets (Behnke, 1967) and during the extremely rapid or “cataclysmic” shortening of Echinosphaeriurn axopodia caused by treatment with Cu2+ or Ni2+ (Roth and Shigenaka, 1970). ”

E. THEASSEMBLYA N D DISASSEMBLY OF LABILE MICROTUBULE ARRAYS: CYTOPLASMIC MICROTUBULES Many cells contain microtubule arrays during interphase as well as during mitosis. Like mitotic tubules, cytoplasmic tubules are labile and subject to disassembly by cold, high pressure, and treatment with antimitotic drugs such as colchicine and vinblastine. Cytoplasmic microtubules play diverse roles in different cells, including formation and/or maintenance of cell shape and involvement in secretion, transport, and other intracellular movement (see Roberts, 1974; Stephens and Edds, 1976). A special widely studied class of cytoplasmic microtubules are neurotubules; because of its quantify and ease of isolation neurotubulin has been the basis of most biochemical studies of tubulin (see Sections I1 A,B). An early study by Gibbins er al. (1969) on the function and regulation of cytoplasmic microtubules concerned formation of the primary mesenchyme in embryos of the sea urchin Arbacia puncrularu. They suggested that control over


31

the distribution of microtubules (hence over the developmental sequence of cell shape changes) might consist of sequential “activation and repression of nucleating sites. In cells of early blastulae, the cytoplasmic microtubules originated from two or three “satellites,” electron-dense masses arranged around the basal body at the apical part of the cell, from which point they diverged outward and extended downward parallel to the cell periphery. In later-stage blastulae, microtubules in presumptive mesenchyme cells (which round up and migrate into the blastocoel) appeared randomly oriented. However, at this point is illustrated one of the difficulties of electron microscope studies, namely, that they give static pictures of dynamic events with concomitant difficulties in interpretation. The basal bodies in the early blastula cells observed bore cilia, but later studies have shown that the micromeres which arise at the 16-cell stage and ultimately become the primary mesenchyme do not become ciliated (Okazaki, 1975; Raff et a l . , 1975). As Gibbins and co-workers pointed out, it was difficult to locate these cells in thin sections in early blastulae; thus it was not certain that the two kinds of ectodermal cell microtubule arrangements in fact represented sequential alterations in the presumptive mesenchyme cells. In later stages, the sequence could be clearly followed, since the differentiating cells could be more easily identified. In newly formed mesenchyme cells microtubules radiated from the cell center; in some cases a few microtubules appeared to contact the wall of the centriole. During migration and formation of the cable syncytium the microtubules in both the pseudopodia and stalks appeared in arrays parallel to the direction of extension, while in the cell body the microtubules appeared to extend from dense masses, or satellites, near the nuclear envelope (but did not contact the centrioles). Tihey and Gibbins (1969) further studied these events by treating ernbryos with colchicine, deuterium oxide, or increased hydrostatic pressure; they concluded that the changes in microtubule orientation were important in developing but not in maintaining the changes in cell shape. Tilney and Goddard (1970) confirmed that the electron-dense satellites in ectodermal cells of early blastulae in fact represented sites of initiation of microtubule assembly. When they decreased the temperature to 0”C, the microtubules disassembled but the satellite structures remained; on rewarming, microtubules arose at the satellites. Warren (1974) followed the involvement of microtubules in the morphogenesis of developing muscle cells in the tail of Rana pipiens tadpoles by electron microscope observation of serial sections through portions of developing myoblasts. He observed that during development of these cells there was a shift in microtubule organization from radial arrays early in development to parallel arrays in differentiated cells. In mesenchyme cells and in some premyoblast cells the microtubules radiated from amorphous electron-dense centriolar satellites, whereas in more mature myoblasts and myotubes the microtubules were arranged along the length of the cell and were no longer focused on the cell center. The site ”

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ELIZABETH C. RAFF

of origin of microtubules in mature cells was unclear. Warren observed many apparently abrupt terminations of microtubules in serial sections; some ends of microtubules were free in the cytoplasm, but frequently these ends were associated with various cell membranes and in a few cases with amorphous, electron-dense material. Recently, the ability to observe the process of formation and disappearance of both mitotic and cytoplasmic microtubules has been greatly enhanced by the use of indirect immunofluorescence. Briefly, specific antibodies are prepared to purified tubulin. Cells to be examined are then fixed on slides and reacted with the antitubulin; the areas andor structures in the cell which have bound the antitubulin molecules are subsequently amplified as well as visualized by treatment with fluorescence-labeled antibodies from a second species (often sheep or goat) raised against immunoglobulins of the species (usually rabbit) in which the antitubulin antibodies were raised. A common feature of these experiments is that the antitubulin antibodies bind to microtubules from a wide variety of heterologous species. Antibodies prepared against mammalian brain tubulin have been shown to decorate microtubules and tubulin-containing structures in several mammalian and vertebrate cell types (Brinkley et a / . , 1975a,b,, 1976; Edelman and Yahara, 1976; Frankel, 1976; Fujiwara and Pollard, 1978; Fuller and Brinkley, 1976; Fuller et al., 1975a; Osborn and Weber, 1976a,b, 1977; Schliwa et al., 1978; Weber, 1976) and in invertebrates, plants, and protozoa (Franke et al., 1977; Weber et al., 1977b). Similar results have been obtained with antisera against sea urchin sperm tail tubulin (Weber, 1975, 1976; Weber et a/., 1975a,b), sea urchin egg tubulin (Sato et al., 1976), and tissue culture cell tubulin (Isenberg et a/., 1977). The structures stained were shown to be authentic microtubules by the use of known microtubule-depolymerizing agents such as cold, colchcine, and vinblastine; the binding appears to be specific for tubulin with the possible exception of some nuclear fluorescence, the nature of which is unclear, observed in only a few cell lines. As shown in Fig. 6, the most striking result of these studies is that not only is the mitotic apparatus stained, giving an appearance at each stage of mitosis virtually identical to that obtained by birefringence studies, but that an elaborate network or cytoskeleton of microtubules exists in nearly all the interphase cells examined. This cytoplasmic microtubule network disappears during prophase and is completely absent during mitosis, when most of the fluorescence appears in the mitotic apparatus, although in some cell lines background fluorescence increases slightly over the interphase level. The cytoplasmic microtubule complex then reappears after disassembly of the mitotic apparatus. Whether the same pool of tubulin subunits is utilized in both cytoplasmic and mitotic tubules is an interesting but unresolved question. The reciprocal appearance and disappearance of the two sets of microtubules have been taken as implicit evidence that this may be so, but there is no direct unequivocal biochemical proof. Certainly the

Fic;. 6. Microtubules in mouse 3T3 cells in culture visualized by indirect immunofluorescence with a monospecific antibody against tubulin. (a) Interphase cell, showing the cytoplasmic microtubule network. ~ 7 6 5 (b) . Interphase cell after treatment with colchicine ( I pLp/nil) for I hour. x765. In both (a) and (b) the microtubule-organizing structure in the cell center is clearly visible. (c) Mitotic cell. showing astral and spindle tubules. X765. Reprinted from Osborn and Weber (1976b). with permission.

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ELIZABETH C. RAFF

organizational events determining the pattern and orientation of assembly appear to be separate. The dismantling of cytoplasniic microtubules often involves the loss of asymmetry (i.e., the rounding up of cells before division); this may simply ensure proper formation of daughter cells. The cytoplasmic microtubule arrays revealed by immunofluorescence staining appear to emanate from a small number of sites in the cell center. Osborn and Weber (1976a,b) monitored elaboration of the cytoplasmic microtubule network in mouse 3T3 cells in culture recovering from treatment with cold or colchicine and during the process of spreading and attaching to glass substrates. They obtained clear evidence that microtubules arose from one or two discrete sites in the center of the cytoplasm and grew in a polar manner toward the cell periphery (Fig. 6a,b). When the microtubules first approached the plasma membrane, they often appeared to contact or at least approach it closely and in some cases appeared to “stretch” it. The microtubule-organizing structure close to the nucleus was visualized as a fluorescence-staining cylinder about 3 pm long, itself exhibiting polarity in that one end was above the plane of the microtubules, which arose directly from the bottom end of the cylinder. This structure persisted in cells in which the cyotplasmic microtubules were depolymerized by cold or drugs. Its relation, if any, to centrioles is unclear as yet. Osborn and Weber suggested that two types of regulation must be involved in the assembly of cytoplasmic microtubules: (1) positive regulation, in which assembly takes place with specific timing and orientation from the organizing site, and (2) negative regulation, in which assembly outside the specific pathway is inhibited, preventing the formation of random or unoriented microtubules. Frankel (1976) observed the outgrowth of microtubules in cultured mouse macrophages and fibroblasts. Macrophages showed a single microtubule-organizing center which appeared as a fluorescent ring with a dark center from which microtubules extended radially in an asterlike pattern; fibroblasts showed a typical complex network of microtubules. At initial stages of regrowth after microtubule depolymerization caused by cold or drugs, microtubules in fibroblasts grew out toward the cell membrane from one to three foci near the cell center. The cytoplasmic microtubule network has also been visualized at the electron microscope level. Two groups have employed the immunoperoxidase method, in which fixed cells are treated sequentially with antitubulins, then antiimmunoglobulins, and then a complex of peroxidase and antiperoxidase; after incubation with a peroxidase substrate, the reaction sites are ultimately made visible with electron microscopy by the deposition of osmium. Using this method DeMey et al. (1976) and DeBrabander et al., (1977ii,b) observed a typical network of cytoplasmic microtubules radiating from the cell center toward the cell periphery in mouse embryo cells in culture. These investigators interpreted the diffuse staining observed in the cytoplasm to indicate the presence of tubulin subunits;


35

this background staining increased when cytoplasmic tubules were disassembled by colchicine treatment and decreased when vinblastine crystals, which stained heavily, were formed. Because the staining was evenly distributed, they suggested that no gross compartmentalization of tubulin subunits occurs in these cells. This group also reported that this method could be used at the light microscope level (DeBrabander et al., 1977b). In rat kangaroo cells in culture, Pepper and Brinkley (1977) observed staining in kinetochores, electron-dense amorphous pericentriolar material, and small viruslike particles associated with centrioles, in addition to that in centrioles and the mitotic spindle. Thus tubulin may be a component of microtubule-organizing centers. The peroxidase staining was uniform throughout the kinetochore, even though microtubules appeared to be associated only with the outer layer. Eckert and Snyder (1978) combined the indirect immunofluorescence technique with high-voltage electron microscopy by employing antibodies raised against glutaraldehyde-treated tubulin, confirming the identity of the networks observed at the light microscope level with microtubules. Immunofluorescence studies of the cytoplasmic microtubule network have raised the possibility that it may be altered in transformed cells. Brinkley et al. (1975a,b; 1976) reported that, whereas in several lines of normal fibroblastlike mammalian cells in culture they observed a typical complex network of cytoplasmic microtubules radiating from one or two densely staining areas near the nucleus, cells transformed by viruses, chemically or spontaneously, contained either randomly oriented or very few microtubules. However, during mitosis there was no detectable difference in the microtubule patterns between transformed or nontransformed cells. This group recently repeated these observations using hybrids of transformed mouse cells and normal human fibroblasts; only cells showing normal growth patterns exhibited the complete microtubule network, while cells showing intermediate or transformed growth patterns all showed diminished or absent microtubule networks (Miller et al., 1977). Similarly, Edelman and Yahara (1976) observed distinct microtubule networks in normal cells, whereas antitubulin staining in transformed cells was diffuse and unstructured. Both patterns could be observed in the same cells: chicken fibroblasts infected with a temperature-sensitive Rous sarcoma virus and grown at permissive temperatures showed the typical transformed morphology and diffuse pattern of antitubulin staining; at restrictive temperatures the typical cytoplasmic microtubule network, and normal cell shape, reappeared within 1 or 2 hours, However, Osborn and Weber (1977) demonstrated cytoplasmic microtubule networks in several lines of transformed mammalian cells in culture and suggested that the rounded morphology of transformed cells makes the observation of microtubules by indirect immunofluorescence technically difficult. DeMey et al. (1978) also observed microtubule networks in transformed cells at

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ELIZABETH C. RAFF

both the light and electron microscope levels using the immunoperoxidase technique; in some tumor cell lines their electron microscope observations revealed microtubule networks which were poorly visualized at the light microscope level. 11. The Dance: Experimental Dissections of Microtubule Assembly in Vivo

It is difficult to separate the observation of a biological system from experimentation; the very procedure of microscopic observation itself, after all, involves meddling with the system. Also, many of the observations discussed in Section I were made on systems in which microtubule assembly or disassembly had been induced by altering normal physiological conditions (i.e., treatment with cold, pressure, drugs, and so on). Nevertheless, the basic rationale behind these studies was to describe the processes of microtubule assembly as they actually normally occur in cells. The work to be discussed now is more clearly experimental in motive; that is, it is aimed at defining the mechanisms which govern microtubule assembly in vivo by altering andor isolating the components of the microtubule assembly process. A real difficulty with experiments on highly purified preparations is that they may not accurately reflect physiological conditions. However, experiments performed in sifu or on partly purified preparations have the reciprocal drawback, always present in examining complex systems, of the difficulty of sorting out specific from nonspecific effects. A. THEBIOCHEMISTRY OF TUBULIN, THE STRUCTURAL MICROTUBULE PROTEIN

Tubulin was first identified as the major component of microtubules by observation of its binding of the antimitotic drug colchicine (Borisy and Taylor, 1967a,b; Shelanski and Taylor, 1967, 1968; Weisenberg e f al., 1968; L. Wilson, 1970; Wilson and Meza, 1973). The recent reviews by Snyder and McIntosh (1976) and Stephens and Edds (1976) contain good summaries of the chemistry of tubulin and the structure of microtubules. The functional subunit in the assembly of microtubules is a 110,000-molecular-weight heterodimer of the two tubulin monomers (Luduena et a l . , 1975, 1977); a and /3 tubulin are related peptides with similar molecular weights and electrophoretic properties, but different primary structures (Luduena and Woodward, 1973, 1975) coded for by separate genes (Bryan et ui., 1978). Tubulin synthesis may accompany the formation of a microtubule-containing organelle but, as discussed above in connection with studies of ciliagenesis, the synthesis of tubulin only rarely appears to be the controlling point for microtubule assembly. Rather, in general it appears that most cells maintain substantial tubulin pools. For example, many developing embryos maintain a relatively constant level of tubulin throughout early development: the axolotl (Raff, 1977;


37

Raff and Raff, 1978); Drosophila (Green et al., 1975); the sea urchin (reviewed by Raff et al., 1975; see also the discussion of ciliagenesis, above); and Spisula (Bumside et al., 1973). Modulation of tubulin levels occurs, as indicated by the changes in relative rates of tubulin synthesis observed during the cell cycle in mammalian cells in culture (Klevecz and Forrest, 1975; Lawrence and Wheatley, 1975), in sea urchin embryos (Rubin et al., 1976), and in Chlamydomonas (Pipemo and Luck, 1977). Such changes have not been found to be involved in the regulation of cell division, however. Given that cells contain reserves of tubulin which may be assembled into microtubules, to date it has been difficult to define which properties of the tubulin monomers serve regulatory functions in vivo. First, binding sites are present on tubulin molecules for several small molecules of which the most important appear to be: 1. Drugs such as colchicine, vinblastine, and podophyllotoxin, and so on, which act as mitotic poisons (see, for example, Bhattacharyya and Wolff, 1977; Bryan, 1972; Cortese et al., 1977; Garland and Teller, 1975; Harrisson et al., 1976; Kelleher, 1977; Lee et al., 1975; McClure and Paulson, 1977; Owellen et al., 1972; Pfeffer el nl., 1976a,b; Schmitt and Atlas, 1976; L. Wilson, 1970, 1975; Wilson et al., 1974, 1975a,b). 2. Guanine nucleotides (Berry and Shelanski, 1972; Bryan, 1972; Shelanski and Taylor, 1968; Stephens et a l . , 1967; Weisenberg et al., 1968). 3. Metal ions, including magnesium (Lee and Timasheff, 1975, 1978; Olmsted and Borisy, 1975), calcium (Hayashi and Matsumura, 1975; Rosenfeld et al., 1976; Solomon, 1976, 1977), and lithium (Bhattacharyya and Wolfe, 1976a). The binding of antimitotic drugs is the most widely used biochemical tool for tubulin characterization. How the drug-binding sites participate in normal function is unknown. The binding of nucleotides and magnesium is required for microtubule assembly in v i m , but it is not clear if they serve a regulatory function. Calcium is an inhibitor of microtubule assembly in vitro and is a good candidate for an in vivo regulator. These are discussed more fully in following sections. Several posttranslational modifications of tubulin monomers have been observed. Unfortunately, evidence on their possible functions in vivo is incomplete and sometimes conflicting. First, tubulin has been reported to be a glycoprotein (Margolis er al., 1972). Feit and Shelanski (1975) observed that tubulin from particulate fractions but not from soluble fractions was glycosylated after incorporation of gly~osamine-'~C into mouse brain in vivo. Second, Eipper (1969, 1974, 1975) showed that tubulin from rat brain is phosphorylated at a single serine residue in the p chain; she also observed that the labeling patterns of 32P

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ELIZABETH C. RAFF

incorporation into tubulin in brain slices from newborn rats were different from the patterns in adult brain. Phosphorylation of tubulin has also been reported in chick brain (Lagnado and Kirazov, 1975). Piras and Piras (1975) reported that the extent of phosphorylation of tubulin from HeLa cells varied during the cell cycle, but phosphorylation of tubulin could not be detected at all in Chinese hamster ovary (CHO) cells in culture (Rubin and Weiss, 1975) or in normal or differentiating neuroblastoma cells in culture (Solomon et al., 1976). Tubulin was earlier thought to possess intrinsic protein kinase activity, but it has been shown that this activity, although sometimes copurifying with brain tubulin assembled in virro, is separable (Rappaport et al., 1976; Sandoval and Cuatrecasas, 1976a). Finally, it has been shown that tyrosine is covalently linked to the carboxyl terminal residue of the a subunit of brain tubulin by a specific posttranslational enzymic reaction (Arce et al., 1975; Argarana et al., 1977; Barra et al., 1974; Raybin and Flavin, 1975, 1977a). Halleck et al. (1977) reported that the tyrosine residue was released under conditions which promoted assembly in vitro of microtubules from rat brain fractions. However, Raybin and Flavin (1977b) found that only some brain tubulin purified by assembly is tyrosylated and that tubulin assembled just as well in vitro with or without carboxyl terminal tyrosine. Furthermore, while they found tubulin-tyrosylating activity in other rat tissues besides brain, tyrosylation of tubulin appeared to occur primarily on tubulin found in insoluble fractions in cultured neuroblastoma cells and did not occur at all in various invertebrates they examined. Tubulin is evolutionarily a highly conserved protein. Fulton et al. (1971) originally showed by cross-reactivity of antibodies against tubulin from sperm tails of the sea urchin A . punctulata that flagellar and mitotic microtubules from several species of sea urchins and a sand dollar were extremely similar, although not identical. The immunofluorescence studies discussed in Section I, exploiting the cross-reactivity of antitubulin antibodies with diverse heterologous microtubules, also well illustrate this point. This evolutionary constancy of both the morphology of microtubules and the structure of the tubulin subunits has led many workers to place a quite reasonable implicit emphasis on the similarities of tubulins from different organisms. It has only recently been appreciated that a major means of regulation of tubulin function may lie in small differences in the tubulin molecules even of a single organism. That is, what has been thought of as the “tubulin pool” in a cell may actually consist of multiple subpools in which the tubulin subunits are heterogeneous either because of actual differences in primary structure or secondary posttranslational modifications (or both). Of course differences in the behavior of microtubules have been known almost as long as microtubules have been recognized. Behnke and Forer (1967) originally classified microtubules into four groups based on stability to fixation and solubilization, the most labile being those in the mitotic apparatus and the most stable those in the axoneme of flagella or cilia. It is still not clear whether these


39

differences reflect differences in the biochemistry of the composite tubulins or whether they depend on the cellular environment of the various tubules. It is very obvious that the most stable microtubules are those which are part of more complex structures; associated structures such as arms, doublet or triplet tubules, spokes, linkers, and so on, may indicate the presence of binding sites not present on other microtubules. How such sites are specified has not been determined. Stephens (1975) has suggested that there may be differences in the tubulins comprising the A and B tubules in the outer doublet tubules of sea urchin sperm flagella. He obtained in v i m reassembly of tubulin isolated from A tubules into single tubules, but tubulin from B tubules formed only sheets or ribbons, possibly because the preferred mode of self-association of the tubulin which in vivo forms the doublet portion of the tubule precludes formation of a single tubule; however, no doublets formed in mixed solutions of tubulin from A and B tubules. The biochemical evidence was not definitive. Large cyanogen bromide cleavage peptides were similar for both Q and /3 tubulins from A and B tubules. There were some differences in the amino acid profiles of the different subunits, but total amino acid analysis of such large peptides is not a sensitive technique and the differences observed were slight. More recently Stephens (1977b) obtained evidence from radioactive labeling experiments that the tubulin pools for the A and B subfibers of the outer nine doublet tubules of sea urchin embryo cilia are separate. Wilson et al. (1975a) suggested that several populations of microtubules exist in brain because of the differential sensitivities they observed in preparations of brain tubulin to the antimitotic agent griseofulvin. Feit er al. (1977) found that two-dimensional gel electrophoresis resolved both a- and Ptubulin subunits from chick, adult mouse, and adult bovine brain, as well as from mouse neuroblastoma cells in culture, into multiple bands. Berkowitz et al. (1977) and Bryan er af. (1978) found that this method resolved the a- but not the P-tubulin subunit from calf and chick brain, respectively, into two bands. Similarly, Raybin and Flavin ( 1 977b) and Lu and Elzinga ( 1 977) found that the a- but not the P-tubulin subunit from rat and calf brain, respectively, separated into two peaks on hydroxyapatite chromatography; the biochemical basis for the separation was not clear but apparently did not involve carboxy terminal tyrosylation. Kobayashi and Mohri (1977) obtained a single peak on hydroxyapatite columns for both aand Ptubulin subunits from starfish sperm flagella, but both subunits subsequently yielded multiple bands on isoelectrofocusing gels. In the absence of further biochemical characterization, resolution into multiple bands, particularly on isoelectrofocusing gels, must be interpreted with caution, but there are certainly strong indications that microheterogeneity exists in tubulins from a single tissue or cell. There is good evidence for heterogeneity among tubulins from different tissues or structures in the same organism. Kowit and Fulton (1974a,b) and Fulton and

40

ELIZABETH C. ICAFF

Kowit (1975) showed that, during transformation from the ameba to the flagellate state in N. gruberi, essentially all the tubulin utilized in assembly of the flagella axoneme is synthesized de novo. This is not because the amebas lack sufficient tubulin; tubulin represents nearly 12% of the total cell protein, whereas the flagellar outer doublets comprise only 0.15% of the total protein. Rather, the tubulin synthesized appears to be a unique molecule; antibodies prepared against flagella tubulin did not cross-react with tubulin present in the amebas, or indeed with soluble tubulin present in the flagellates. Fulton and Simpson (1976) therefore speculated that in general, separate genes code for tubulins designed for specific functions. Pfeffer et al. (1976a,b) found that tubulin from sea urchin eggs had a binding constant for colchicine 10-fold lower than that from solubilized sea urchin sperm tail outer doublets (which had a binding constant similar to that observed for brain tubulin). Bibring et al. (1976) also examined tubulins from different sea urchin tissues. In electrophoresis on polyacrylamide gels containing both sodium dodecyl sulfate (SDS) and urea the a subunits of tubulin purified from isolated mitotic apparatus and from the A tubule of cilia, but not from outer doublet tubules of sperm flagella, separated into two bands, whereas in gel systems containing only SDS or only urea, the a subunits from all three migrated similarly, as did the fl subunits in all the gel systems examined. Isoelectrofocusing gels revealed several distinct differences in cyanogen bromide peptides obtained from a tubulin from mitotic apparatus and flagella outer doublets, although the majority of bands were the same. Finally, we have recently observed that tubulins from adult tissues (brain and testis) of the Mexican axolotl and the salamander Necturus differed in electrophoretic properties, although not in colchicine-binding properties, from the tubulin present in eggs and embryos (Raff and Raff, 1978). Furthermore, specific limited proteolysis with both chymotrypsin and a Staphlococcus protease yielded distinctly different peptide patterns from axolotl egg and testis tubulins; the microheterogeneity was more marked in the a than in the fl subunits.

B. MICROTUBULE ASSEMBLY in Vitro: POSSIBLE REGULATORY FACTORS Because microtubule assembly in vivo is complex and difficult to deal with directly, much effort has been devoted to delineation of the process as it occurs in vitro. Weisenberg (1972b) first reported that microtubules could be reconstituted in vitro from supernatants of brain homogenates. Microtubule assembly occurs under essentially physiological conditions at 37"C, slightly acid or neutral pH, and moderate ionic strength, and requires the presence of Mg2+ and GTP and the absence of Ca'+. The microtubules that form in vitro have the same appearance as cytoplasmic microtubules in vivo and in most cases are subject to disassembly by the same agents, namely, cold, colchicine and other antimitotic drugs, and increased pressure. The assembly process consists of two separable


41

steps, nucleation or initiation, and elongation. Microtubule assembly in vitro has been extensively reviewed (see, for example, Borisy et al., 1975, 1976; Olmsted et al., 1974; Penningroth et al., 1976; Stephens and Edds, 1976). The majority of the studies of microtubule assembly in vitro have been performed on tubulin isolated from brain; this system is ideal because tubulin constitutes up to 20% of total brain protein. However, assembly of microtubules in vitro has also been demonstrated with tubulin isolated from developing embryos of Drosophila melanogaster (Green et al., 1975, 1979), marine invertebrate eggs (Kuriyama, 1977), and a variety of nonneural mammalian cell types (Barnes et al., 1975; Castle and Crawford, 1975; Doenges et al., 1977; Nagle et al., 1977; Weber et al., 1977a; Wiche and Cole, 1976; Wiche et al., 1977a,b). Several studies have been aimed at defining which amino acid residues of the tubulin subunits participate in the assembly process. Modification of tyrosine (Gaskin and Gethner, 1976) and histidine residues (Lee et al., 1976) prevents assembly. Several laboratories have proposed that sulfhydryl groups are involved in the control of tubulin function. The number of free sulfhydryl groups measured varies somewhat depending on the conditions used in preparing the tubulin but is usually reported to be seven per 55,000-molecular-weight monomer, of which two are required for assembly (Kuriyama and Sakai, 1974; Mellon and Rebhun, 1976a,b; Nishida and Kobayashi, 1977; Wallin et al., 1977). The details of the ionic requirements reported for the assembly of microtubules in vitro vary considerably, depending on the source and method of preparation of the tubulin and on other buffer components. Thus the standard assembly conditions require 0.5 mM magnesium (Shelanski ef a/., 1973; Weisenberg, 1972b); concentrations of 10 mM or above may also stimulate assembly (see below), but under other conditions inhibit assembly (Olmsted and Borisy, 1975) or cause aggregation and precipitation of tubulin (Weisenberg and Timasheff, 1970). Calcium is a potent inhibitor of microtubule assembly in vitro, but whether inhibition occurs at millimolar or micromolar concentrations depends on the magnesium concentration (Olmsted, 1976; Rosenfeld et al., 1976). Thus it has been difficult to determine which buffer conditions used in vitro represent conditions in vivo. The second requirement for microtubule assembly in vitro, that for guanine nucleotides, is also complex. Tubulin-nucleotide interactions have been reviewed by Jacobs (1975), Olmsted (1976), Weisenberg (1975), and Weisenberg et al. (1976b). Briefly, tubulin contains at least two binding sites for guanine nucleotides which differ in the extent of exchange with exogenous nucleotides. The role and function of nucleotides at these two sites is incompletely understood as yet, and the data thus give a somewhat conflicting picture. As Weisenberg et al. (1976a,b) have pointed out, the state of bound guanine nucleotide is highly sensitive to the conditions under which tubulin and microtubules are observed. The more readily exchangeable site, which Geahlen and Haley (1977) have shown to be on the /3 subunit, appears to be most important in microtubule

42

ELIZABETH C. ICAFF

polymerization. GTP or GDP (which is subsequently transphosphorylated to GTP) is bound at this site and is hydrolyzed under conditions which also allow polymerization (David-Pfeuty et al., 1977), but it is not clear if hydrolysis is a requirement for, or merely concomitant with, polymerization; in some studies it was found that hydrolyzable GTP was necessary to obtain polymerization (Maccioni and Seeds, 1977; Olmsted and Borisy, 1975), while in other studies nonhydrolyzable analogs of GTP supported polymerization (Arai and Kaziro, 1977; Penningroth and Kirschner, 1977; Penningroth et al., 1976; Weisenberg and Deery, 1976). Assembly was also reported in the absence of added nucleotides when glycerol or sucrose, which stabilize microtubules, was present in the assembly buffer (Shelanski et al., 1973). These results may be reconciled by the finding of Penningroth and Kirschner (1978) that the nucleotide specificity at this site is broad and that only substoichiometric amounts of bound trinucleotide are required for polymerization to occur. Sandoval et al. (1977) found that purified tubulin not only asembled more efficiently in the presence of a hydrolyzable GTP analog (guanylyl 5'-methylene diphosphonate, GMP-CH,-P-P), but that the resultant tubules were also more stable than those formed in the presence of GTP. This is consistent with the data of Arai and Kaziro (1977) and Penningroth and Kirschner (1977), who both concluded that binding of GTP at this site induces conformational changes favoring assembly. Jacobs and Caplow (1976), however, concluded that the role of GTP at this site is not as an allosteric effector. Zeeburg et al. (1977) reported that the GDP resulting at this site may also be cleaved, forming a covalent GMP-protein bond, which they suggested as a possible regulatory mechanism. GTP is also bound at the less readily exchangeable site; hydrolysis of this moiety has generally not been observed (Penningroth and Kirschner, 1977; Spiegelman et al., 1977; Weisenberg et al., 1976b) but has been reported to occur (MacNeal and Punch, 1977). Spiegelman et al. (1977) have suggested that the nucleotide bound at this site, unlike that at the exchangeable site, may be a stable structural component or cofactor and is not involved in the control of assembly or disassembly. In the initial studies of microtubule assembly in vitro, several laboratories observed that both crude preparations of brain tubulin and tubulin resolubilized from microtubules assembled in vitru could be separated by differential centrifugation or molecular seive chromatography into two fractions, both composed primarily of tubulin, one of which was competent to initiate self-assembly and one of which was not (Borisy and Olmsted, 1972; Borisy et al., 1974, 1975, 1976; Bryan, 1976; Erickson, 1974b; Kirschner and Williams, 1974; Kirschner et al., 1974, 1975a,b; Penningroth et al., 1976; Scheele and Borisy, 1976; Shelanski et al., 1973; Weisenberg, 1974; Weisenberg and Rosenfeld, 1975a). One fraction corresponded to the 1 10,000-molecular-weight 6 s tubulin dimer, but the other was composed of high-molecular-weight oligomers of tubulin with sedimentation constants from 18 to 36s. Electron microscopy of the latter frac-

43


tions revealed highly ordered ring-or disklike structures, the exact structure of which depended on the source and conditions of preparation of the tubulin. Although the oligomeric fractions could reversibly break down into 6 s dimeric tubulin and were competent to reassemble into microtubules, the 6s fractions neither formed rings or disks nor reassembled into microtubules unless mixed with the 36s fraction. Many laboratories postulated that these disklike structures represented the initiation step of microtubule formation in vitro. However, this does not appear to be the case. Weisenberg (1974) showed that, although rings were present initially and disappeared during the subsequent course of polymerization, they were not incorporated directly into microtubules but were first broken down into smaller subunits. More recently, in detailed kinetic and electron microscope analyses, Bryan ( I 976) showed that the rings are not required either for nucleation or for elongation of microtubules. The function of these structures remains unclear; they appear to be purely in vitro structures resulting from the experimental manipulation of molecules which have the potentiality for several modes of self-association. Rings and disks are frequently observed to form under conditions (such as in the cold) under which microtubule assembly is inhibited. Certainly no structures resembling them have been observed in electron microscope studies of microtubule systems in vivo. Experiments designed to determine why some brain tubulin preparations were competent to initiate self-assembly into microtubules while others were not, and to define the nature of the rings and disks observed in assembly-competent fractions, brought up another point crucial to the understanding of both the process and the control of microtubule assembly: the possibility of a requirement for factors in addition to tubulin, which might act either stoichiometrically or catalytically to allow initiation andor elongation of microtubules. It was observed that microtubules formed in vitro frequently contained proteins in addition to tubulin, particularly a series of proteins in the molecular weight range from 200,000 to 350,000 which copurified with tubulin through several cycles of assembly and disassembly (for example, see Borisy et al., 1974; Bums and Pollard, 1974; Dentler et ul., 1975; Erickson, 1974b; Gaskin et al., 1974b; Haga and Kurokawa, 1975; Keates and Hall, 1975; Kirschner e t a / . , 1974; Kuriyama, 1975; Sloboda et a/., 1975, 1976a). These proteins together with a more recently identified group of proteins of lower molecular weight, collectively termed tau factor (Weingarten et ul., 1975), have been identified as such microtubule assembly-promoting factors. Amos (1977b) has presented an excellent short summary of this work. Tubulin fractions competent to self-assemble appear to be those also containing these proteins, which collectively have been termed microtubule-associated proteins (MAPs). In recent literature, tubulin, together with MAPs, has been referred to as microtubule protein(s). [A word of caution is in order here, since in earlier literature the latter term, “microtubule protein(s), was used synony”

44

ELIZABETH C. RAFF

mously with “tubulin. ”1 Both the number and nature of the MAPs and the details of how they affect microtubule assembly vary in different experiments. Most studies of MAPs indicate that they act stoichiometrically to promote the assembly of microtubules in vitro and are more important in the initiation of assembly than in microtubule elongation. Whether they are regulatory proteins for microtubule assembly in vivo is currently a much-debated question. Recent work has emphasized the separation of pure 6s tubulin from MAPs by ion-exchange chromatography: either by anion exchange, in which case tubulin is retained on the column and the MAPs are eluted under low salt conditions, or by cation exchange, in which case the MAPs are retained and tubulin is eluted first. Under standard assembly conditions, tubulin so purified does not initiate extensive self-assembly into microtubules, but does so if the MAP fraction is added back. Murphy and Borisy (1975) and Murphy ef af. (1977a,b) examined the assembly of porcine brain tubulin separated from MAPs by DEAE-Sephadex chromatography. Of about 35 nontubulin proteins observable after two cycles of assembly and disassembly only a few bound to microtubules with high affinity and remained at constant stoichiometry through further cycles of assembly. In their preparations the main component which stimulated assembly was the high-molecular-weight proteins, but other stimulating proteins, including the tau group, also appeared to be present. At 5°C the addition of MAPs to the purified tubulin promoted the formation of ring structures; recent work by this group has shown that the type of oligomeric structure formed in the cold is a function not only of solution conditions such as pH and ionic strength but also of which species of MAP is associated with tubulin (Marcum and Borisy, 1978a,b; Scheele and Borisy, 1978; Vallee and Borisy, 1978). At 37”C, rapid assembly into microtubules occurred; while MAPs stimulated both initiation and elongation, they were required only for the former. Murphy et al. (1977a) observed that the stimulation of microtubule assembly by MAPs proceeded with sigmoidal kinetics, which they interpreted to indicate that stimulation of elongation occurred by reduction of the rate of depolymerization, thus shifting the equilibrium to favor assembly. In contrast to models involving the incorporation of MAPs into tubules during assembly they suggested that MAPs bind to sites which become available only after polymerization, thereby stabilizing the microtubule. Some evidence consistent with this idea has been reported by Meier and Jgrgensen (1977), who found that antibodies prepared against intact microtubules assembled from rat brain tubulin in vitro bound to intact microtubules and to MAPs (which in their preparations included tau factor and a 135,000molecular-weight component) but not to soluble 6s tubulin dimers. Sloboda et af. (1976b) reported similar results with bovine brain tubulin purified by chromatography on phosphocellulose; they found that, as the ratio between the amount of high-molecular-weight MAPs and tubulin was increased, a greater number of tubules were initiated and at a faster rate, and in addition the


45

total mass of microtubules assembled increased; thus in this system MAPs were required for both nucleation and elongation. In contrast to these results, Weingarten et al. (1975) did not observe significant amounts of high-molecular-weight MAPs in their preparations of microtubules polymerized in virro from porcine brain, but found that tau factor, a group of proteins with molecular weights similar to that of tubulin, could be separated from tubulin by phosphocellulose chromatography. These proteins appeared to be incorporated stoichiometrically into microtubules and were required not only for the initiation of self-assembly of purified tubulin but also, in lower amounts, for its addition to preformed microtubule seeds (Witman er ul., 1976). Penningroth et a / . (1976) found that crude preparations of tau factor could be separated into two active fractions, and Cleveland et al. (1977a,b) recently reported that purified tau factor accounted for two-thirds of the total assemblystimulating activity in their MAP preparations and consisted of four closely related heat-stable proteins with apparent molecular weights between 55,000 and 62,000. The remaining microtubule assembly-stimulating activity in their MAP preparations was of much lower specific activity and was found in a fraction containing a spectrum of high- and low-molecular-weight proteins. These investigators suggested that tau proteins function by binding several tubulin molecules per molecule, thus facilitating the formation of longitudinal filaments, hence microtubule assembly. Fellous et ul. ( 1977) investigated the microtubule assembly-stimulating activity in MAP fractions from rat brain and found that in their preparations it resided entirely in the low-molecular-weight material. The association of nontubulin proteins with microtubules in vivo was inferred by Kirkpatrick et ul. (1970), who observed high-molecular-weight components in preparations of intact microtubules isolated from brain. Burton and Femandez (1973) observed “fuzzy” projections evenly spaced along the length of the tubule walls of axonal neurotubules in situ. Several workers subsequently observed similar projections on the surfaces of microtubules isolated intact from blood platelets and brain (Behnke, 1975), and on tubules assembled in vitro from combined purified tubulin plus MAP fractions (Amos, 1977a; Dentler et al., 1975; Murphy and Borisy, 1975; Sloboda et al., 1976a). These projections were interpreted as evidence that the high-molecular-weight MAPs were incorporated as structural components in the microtubule walls. Some microtubules formed in vitro are clearly smooth-walled, depending on the conditions under which they are assembled (Bloodgood and Rosenbaum, 1976; Sloboda et u / . , 1976a), and such projections are absent in most electron micrographs of microtubules in vivo. However, Amos (1977a) has recently reported that whether these projections are seen or not may depend on the methods used for fixation and staining. The similarities between the high-molecular-weight MAPs and dynein, the high-molecular-weight ATPase which constitutes the arms, or projections, on the outer doublet tubules of cilia and flagella (see Gibbons et a / . , 1976) have not

46

ELIZABETH C. RAFF

escaped notice, but a relationship between these proteins, if there is one, has not been established. High-molecular-weight MAPs do not have ATPase activity (Bums and Pollard, 1974; Gaskin et al., 1974b) and appear to differ slightly from dynein in electrophoretic mobility (Murphy and Borisy, 1975). However, Bloodgood and Rosenbaum (1976) observed that the rate of assembly but not the final mass of tubules assembled was enhanced by heterologous high-molecularweight fractions, including dynein, isolated from invertebrate sperm flagella. Such activity of MAPs in heterologous systems is not unique. Bovine brain MAPs have been found to support the assembly of tubulin purified from marine invertebrate eggs by DEAE-Sephadex chromatography (Kuriyama, 1977), and tubulin purified from Drosophilu embryos by phosphocellulose chromatography (Green e f al., 1979). Wiche et al. (1977a) observed that porcine brain tau factor not only stimulated the assembly of rat glial tissue culture cell tubulin but also stabilized the polymerizing ability of the tubulin preparations, which in the absence of added tau factor was lost rapidly after preparation. One possibility which has been considered is that the tau proteins may represent fragments of the high-molecular-weight MAPs. Sloboda et al. (1976b) noted that the high-molecular-weight MAPs were degraded into smaller fragments rather rapidly during storage, with a concomitant loss of their ability to facilitate the initiation of microtubule assembly but retention of their effect on the total amount of assembly. Vallee and Borisy (1977) found that limited trypsin treatment of microtubules assembled in vitro selectively destroyed the highmolecular-weight MAPs and correspondingly removed the lateral projections observed on the tubules. However, the typsin-treated tubulin preparation retained its capacity for self-assembly. These investigators therefore proposed that the assembly-stimulating activity of the high-molecular-weight MAPs actually resided in a small fragment (putatively on the order of 30,000 to 35,000 molecular weight). Against this circumstantial evidence are the recent observations of Cleveland et al. (1977a,b) that peptide maps obtained from tau factor and the high-molecular-weight MAPs are different and of Connolly ef ul. (1978) that antisera against the two species of MAPs do not cross-react. As Amos (1977b) has pointed out, however, since the tau proteins would constitute only 20% of the high-molecular-weight MAP molecules, this matter cannot yet be considered definitively resolved. There is no direct evidence that MAPs are microtubule assembly factors which function in vivo. The strongest indication that this may be so is provided by recent indirect immunofluorescence studies showing that antibodies prepared against purified tau or high-molecular-weight MAP fractions decorate the same cytoplasmic network of fibers decorated by antibodies against tubulin. These studies do not answer the question of how MAPs function but do demonstrate that these proteins are at least localized along with microtubules in cells in vivo. As shown in Fig. 7, Connolly et al. (1977) found that, in mouse embryo fibro-

CONTROL O F MICKOTUBULE ASSEMBLY

47

FIG. 7. Microtubules in mammalian cells in culture visualized by indirect immunofluorescence with antibodies against tuhulin and against MAPs. (a-d) Interphase cells. (e-g) Mitotic cells. (a and e ) Mouse embryo fibroblasts stained with antibody against tubulin. (b and f) Pig embryo fibroblasts stained with antibody against tau factor. (c and g) Rat Ch glial cells stained with antibody against total high-molecular-weight MAPs. (d) Rat C6 glial cells stained with antibody against a-highmolecular-weight MAP. (a) ~ 8 5 0 (; h ) X 6 0 0 ; (c) X500; (d) X850; (e) X775; (f) x 775; (g) x 9 2 5 . Micrographs a, b, d, e and f were kindly provided by J . A. Connolly, Dept. of Anatomy, University of Toronto, Toronto, Ontario. Micrographs c and g reprinted from Connolly c/ ol. (1978) with permission.

48

ELIZABETH C. RAFF

blasts, staining with antitau antibodies was identical in pattern although slightly more diffuse than staining with antitubulin antibodies in both interphase and mitotic cells; in addition, both were colchicine-sensitive; more recently this group demonstrated that in rat glial tissue culture cells, the staining pattern with antibodies prepared against high-molecular-weight MAPs is also similar to that obtained with antitubulin antibodies (Connolly et a l . , 1978). Tubulin, tau factor, and the high-molecular-weight MAPs were antigenically distinct; no crossreactivity was observed among the three antibody preparations. Similar experiments by Sherline and Schiavone (1977, 1978) showed that antibodies prepared against rabbit brain high-molecular-weight MAPs decorated the cytoplasmic microtubule network and the mitotic apparatus in mouse 3T3 cells and cultured neuroblastoma cells. The strongest evidence against the specificity of MAPs in microtubule assembly is the finding reported by several laboratories that ion exchange-purified tubulin can be assembled in their absence if the original assembly conditions described by Weisenberg (1972b) are altered. First, Shelanski et al. (1973) discovered that the inclusion of 4 M glycerol in the assembly buffer both facilitated the rate of assembly and stabilized the resultant microtubules. This modification has been widely employed, and many properties of tubulin assembly in vitro may reflect this stabilization even when glycerol-containing buffers are used only in the initial preparative steps, since glycerol appears to associate tightly with tubulin (Detrich et af., 1976). The minimum (or critical) tubulin concentration necessary for assembly originally observed in brain tubulin preparations also containing MAPs was as low as 0.2 mg/ml (Gaskin et af., 1974a; Olmsted et al., 1974). In the absence of MAPs, purified brain tubulin can be assembled in the presence of glycerol or other stabilizers if the tubulin concentration is on the order of 1 mg/ml (Himes et a l . , 1976, 1977; Lee and Timasheff, 1975; Wehland et af., 1977). Second, since MAPs may be separated from tubulin by virtue of the fact that they bind to anionic resins whereas tubulin does not, several laboratories investigated the effects on microtubule assembly of other polycations such as DEAE dextrans, histones, polylysine, protamine, and RNase A. Such molecules were indeed found to stimulate the polymerization of purified brain tubulin in vitro into either morphologically normal microtubules (Murphy et a l . , 1977b) or double-walled tubules consisting of a normal microtubule with the normal subunit arrangement but with an additional layer of tubulin subunits forming the second outside wall (Erickson, 1976; Erickson and Voter, 1976; Jacobs er a l . , 1975a,b). These observations led Erickson and Voter (1976) to raise the possibility that MAPs have been fortuitously and not specifically copurified with tubulin because of nonspecific electrostatic interactions of these proteins with tubulin. Conversely to the stimulation of polymerization by polycationic substances, Bryan et al. (1975a.b) and Nagle and Bryan (1976) observed that RNA and other


49

polyanions inhibited microtubule assembly in vitro. The inhibition could be overcome by adding back fractions containing MAPs, suggesting that the polyanions inhibited assembly by binding to basic proteins necessary for assembly. These workers proposed that a similar mechanism might prevent spontaneous assembly of tubulin subunits in vivo. Finally, Herzog and Weber (1977) demonstrated that “nonphysiological” buffer components could be avoided altogether. Ion exchange-purified brain tubulin assembled in the absence of MAPs or other factors into morphologically normal microtubules sensitive to cold and colchicine when the magnesium concentration was raised to 10 mM and the tubulin concentration kept above 2.5 mg/ml; whether these conditions reflect those possible in vivo is at the moment controversial. In a detailed thermodynamic study of the effects of solution variables on the assembly of purified brain tubulin in vitro, Lee and Timasheff (1977) found that, depending on the buffer, the critical tubulin concentration ranged from 0.5 to 2 mg/ml; their data were consistent with a simple model of self-nucleated polymerization requiring the binding of magnesium but not requiring additional nontubulin proteins or other factors. Much less information is available on the properties of assembly in vitro of tubulin from nonneural sources, but interesting differences from, as well as similarities to, the properties of brain tubulin have been revealed, demonstrating points at which models for tubulin behavior based on data from brain may not be generally applicable. Doenges et a/. (1977) and Nagle et a / . (1977) found that tubulin from several lines of mammalian cells in culture could be polymerized in vitro in the presence of glycerol. Their preparations did not contain highmolecular-weight MAPs but did have protein components of a lower molecular weight which were removed concomitantly with a loss of polymerizing activity by ion-exchange chromatography. Perhaps most interesting is the fact that preparations of cultured cell tubulin did not normally form ring or disk structures, but did so in the presence of MAPs isolated from brain. We have investigated assembly in vitro from Drosophilu embryos (Green et ul., 1975, 1979). It is worth noting that Drosophilu embryos exhibit one of the highest rates of mitosis known in eukaryotes (as frequently as every 10 minutes) and are thus quite different from brain tissue in their biological demands for microtubules. In the presence of glycerol Drosophilu tubulin polymerizes into normal microtubules assiciated with a small number of nontubulin proteins from 46,000 to 200,000 in molecular weight, similar but not identical to brain MAPs. After purification by phosphacellulose chromatography this tubulin does not spontaneously reassemble alone but is capable of several modes of selfassociation: (1) forming morphologically normal microtubules with normal assembly kinetics in the presence of heterologous MAPS isolated from bovine brain or in the presence of low concentrations of RNase A, (2) very rapidly forming double- and triple-walled microtubules in the presence of 3.3 mg/ml or more

50

ELIZABETH C RAFF

RNase A, and (3) forming macrotubules, single-walled tubules with diameters two to three times that of normal microtubules, in the presence of magnesium concentrations of 16 mM or greater. These are similar to forms seen after vinblastine treatment of rat renal cells in vivo (Tyson and Bulger, 1973) and brain tubulin in vitro (Warfield and Bouck (1974, 1975). Kuriyama (1977) purified echinoderm egg tubulin by DEAE-Sephadex chromatography followed by polymerization in vitro in the presence of glycerol; this tubulin initiated spontaneous self-assembly at concentrations above 2.7 mg/ml; at lower concentrations MAPs isolated from porcine brain or microtubule fragments from cilia, which served as nucleation centers, were necessary in order to obtain polymerization. Maximal assembly occurred at salt concentrations much higher than those at which brain tubulin assembles, but which approach the physiological concentrations in marine eggs. All these experiments illustrate the general difficulty of extrapolating between in vitro and in vivo conditions and reemphasize that the details of tubulin assembly in vitro vary greatly, depending on both the source and also on experimental procedures; furthermore, a variety of tubulin structures may be stabilized in vitro which do not occur in vivo. Erickson and Voter (1976) hypothesized that microtubule assembly in vitro is faciliated by agents (including both MAPs and “nonphysiological polycations) which bind to and cause aggregation of tubulin molecules, thus producing areas of localized elevated tubulin concentrations, at which self-assembly occurs. Such a mechanism may also explain the stimulation of assembly by elevated magnesium concentrations, and of course by elevated tubulin concentrations. They suggested that functional tubulin concentrations in vivo might be on the order of 10-50 mg/ml. It is interesting to note, in light of this hypothesis, that the minimum concentration of soluble tubulin in Drosophila eggs is about 14 mg/ml (Green et al., 1979). Still unexplained is the function of MAPs in vivo; at this point the suggestion by Murphy et al. (1977a) that MAPs in vivo serve to stabilize microtubules seems most attractive. Although the mechanism of initiation of self-assembly of tubulin in vitro remains unknown, a substantial amount of evidence indicates that tubule elongation occurs by the addition of subunits to the ends of the protofilaments composing the tubule wall; the final step in tubule formation is then the folding of the sheet of laterally associated protofilaments into a complete tubule-that is, as has been deduced to occur in vivo, from a C-shaped to a closed, circular cross section (Bryan, 1976; Burton and Himes, 1978; Doenges et al., 1977; Erickson, 1974a; Johnson and Borisy, 1977; Kirschner et al., 1975a; Nagle et al., 1977). An interesting point is the directionality of microtubule elongation, discussed in the following section. A second question is the number of protofilaments which make up the tubules. Single microtubules both in vivo and assembled in v i m have generally been found to consist of 13 protofilaments (Fujiwara and Tilney, 1975; Jones, 1975; LaFountain and Thomas, 1975; Tilney er al., 1973). In ”


51

doublet tubules of flagella and cilia the A tubule consists of 13 protofilaments and the B tubule consists of 10 additional protofilaments attached so that the common wall contains three of the A-tubule protofilaments (for a review of axoneme structure, see Warner, 1974). However, microtubules composed of 12 to 15 protofilaments have been observed in vivo (Burton et al., 1975; Nagano and Suzuki, 1975). Recently Burton and Himes (1978) and Pierson et al. (1978) showed that, although the majority of tubules assembled from bovine brain in vitro contained 13 protofilaments, the number of protofilaments incorporated could vary from 12 to 16 depending upon solution variables such as pH and on the number of cycles of polymerization.

c. THEGROWTHOF MlCRO'lUHULES in

VitrO O N T O

ISOLATED M I C R O T U R U L E -

ORGANIZING C E N T E R S

As is clear from the examples in Section I, microtubules in vivo generally do not appear to arise spontaneously but to grow from distinct, specific sites. This aspect of microtubule polymerization has been reproduced in vitro by several laboratories. A set of experiments on aster formation in homogenates of developing eggs of the surf clam, Spisulu solidissirnu, by Weisenberg (1973) and later by Weisenberg and Rosenfeld (1975a,b) demonstrated that the microtubule-organizing center is a real structure which can be isolated, and that its functionality develops with time. These investigators found that asters formed when homogenates of activated but not of unactivated eggs were warmed after preparation in the cold. Asters did not form in either the pellets or supernatants from low-speed centrifugation of homogenates of activated eggs; however, when a pellet from an activated egg homogenate was mixed with the supernatant from homogenates of either activated or unactivated eggs, asters formed. Thus the pellet was interpreted to contain the organizing center and the supernatant, soluble tubulin. The morphology of the aster formed depended upon the developmental age of the eggs. No asters and few microtubules formed in homogenates of unactivated eggs. Immediately after activation small asters formed in which microtubules arose from a dense central cylindrical structure. At later times large asters formed in which the microtubules arose from electron-dense material surrounding a discernible centriole; someimtes in the latest, largest asters a few microtubules appeared to originate directly from the centriole microtubules. Interestingly, pretreatment with colchicine did not inhibit the formation of a functional microtubule-organizing center, even though no asters formed in homogenates of treated eggs; when the pellet from a treated egg was mixed with a supernatant from an untreated egg, asters formed. Since aster-forming ability was present in activated egg cytoplasm before the appearance of centrioles, it was concluded that centrioles were not directly involved in aster formation.

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ELIZABETH C . RAFF

Experiments in several laboratories have shown that isolated structures such as flagella microtubules and basal bodies will serve as nucleating sites for the polymerization of purified tubulin in vitro. These experiments show that even heterologous tubulin assembles onto these structures, that the soluble tubulin need not be competent to initiate self-assembly, and that the assembly process is polar. Burns and Starling (1974) showed that tubulin in high-speed supernatants of rat brain homogenates, which did not spontaneously reassemble, reassembled when heterologous “seeds” consisting of sea urchin sperm tail fragments were added; conversely, soluble sea urchin tubulin assembled on fragments of rat brain microtubules. Similar results were obtained with solubilized flagella outer doublet tubulin (Kuriyama, 1976). The directionality of microtubule growth was demonstrated in a series of experiments showing that, as illustrated in Fig. 8, assembly of brain tubulin in vitro onto fragments of flagella isolated from Chlamydomonus and sea urchin sperm (Allen and Borisy, 1974; Binder et al., 1975) and basal bodies isolated from Chlumydomonus (Snell et al., 1974; Steams et a l . , 1976), retains the orientation of assembly which occurs in vivo. That is, soluble brain tubulin from high-speed supernatants (which did not initiate self-assembly) assembled primarily at the distal end of both basal bodies and flagella fragments, the rate of elongation depending on the brain tubulin concentration. Some growth of tubules at the proximal ends and at basal body accessory structures such as connecting fibers or rootlets occurred at high tubulin concentrations, but distal assembly was always favored. Microtubules added to both tubules of the central pair but almost entirely to the A tubules of the doublet or triplet microtubules. The formation of doublet or triplet tubules has not yet been observed in v i m . That directionality of growth is an intrinsic property of brain tubulin itself was shown by Dentler et al. (1974). These workers injected chicks intracerebrally with tritiated leucine, isolated labeled brain tubulin, and assembled it in vitro into microtubules; pieces of these microtubules were then incubated with unlabeled tubulin under assembly conditions, and the resulting tubules were exarnined by electron microscope autoradiography . They found that essentially all the radioactivity in the elongated tubules appeared at one end. Recent evidence on the intrinsic polarity of microtubules was provided by Margolis and Wilson (1977, 1978), from experiments on substoichiometric colchicine inhibition of microtubule assembly in vitro and on the rate of exchange of soluble tubulin with microtubules assembled in vitro in the presence and absence of podophyllotoxin, using 3H-GTP as a marker. Their results strongly suggested that, at least in vitro, assembly and disassembly of brain tubulin occur at opposite ends of the microtubule during the steady state. These investigators suggested that possible consequences of this in vivo are (1) an “intrinsic flow” of tubulin subunits through the microtubule, resulting in a specific directionality


53

of microtubule function (e.g., during mitosis, see Margolis e t a / . , 1978), and ( 2 ) that MAPS may function by preventing disassembly. The interpretation of this model for microtubules which have attached ends (i.e., originate at a discrete structural site such as the kinetochore or the distal end of the basal body) seems less clear than for microtubules with two free ends (i.e., self-nucleated tubules assembled in vitro);the model requires that microtubule growth at a site proceed through continued insertion of subunits at the site rather than at the tip, which would then be the disassembly end. This question may relate to the ambiguity of results of experiments discussed in Section I which were designed to determine the point of insertion of tubulin subunits into the growing axoneme of cilia and flagella. At any rate these data are an important consideration as a reminder that the assumption implicit in many studies of microtubule polymerization, that disassembly is the reverse of assembly, may not be entirely valid. Another series of experiments has dealt with the assembly of purified tubulin subunits onto partially purified components of the mitotic apparatus. Cande e t a / . (1974) and lnoue rt ul. (1974) treated lysed mitotic cells and Rebhun et a / . (1974) treated mitotic spindles isolated from Spisulu with solutions containing soluble vertebrate brain tubulin and found that reversible addition of the tubulin to mitotic tubules, accompanied by typical changes in spindle birefringence, occurred. McGill and Brinkley (1975) reported that when HeLa cells arrested in metaphase by colcemid were lysed into solutions containing soluble brain tubulin, microtubules assembled both at the kinetochores and in association with centriole pairs. Milsted et a / . (1977) later exploited the use of brain tubulin solutions to stabilize the mitotic apparatus from blastema stage embryos of D . rnelunoguster so that it could be isolated and examined by scanning electron microscopy. Snyder and Mclntosh (1975) showed that the initiation of new microtubules in mitotic rat kangaroo cells lysed into tubulin solutions took place, as well as elongation of microtubules already present. Microtubules grew directly from the kinetochore but not from the centriole, although they emanated from the surrounding region. These workers suggested the possible involvement of the centriole in microtubule orientation or placement rather than nucleation. Similarly to the Spisulci experiments, this study also demonstrated that the potential for microtubule initiation by the centriolar region is a time-dependent process. “Ripening” occurred between early prophase and late prometaphase, as demonstrated by the increasing number and length of microtubules in asters formed during this period. The assembly of microtubules at the kinetochore was also time-dependent and did not take place before prometaphase. Differential control over the two kinds of microtubules was indicated by the fact that microtubules assembled more readily into asters but kinetochore microtubules were more stable. Pretreatment with colchicine prevented spindle formation but not ripening of the microtubule-organizing center.

54

ELIZABETH C. RAFF

FIG. 8 . The directionality of microtubule assembly i n vifro on isolated organizing structures. Basal bodies (a) and fragments of intact flagella axonemes (b) isolated from Chlomydomonas were incubated with soluble chick brain tubulin. In both cases assembly was clearly preferential at the . from Snell ef al. (1974), with permission. distal end of the isolated structure. (a) ~ 2 8 , 4 6 0Reprinted Copyright 1974 by the American Association for the Advancement of Science. (b) X 13,600. Micrograph kindly provided by L. I. Binder from his Ph.1). Dissertation, Yale University, New Haven, Connecticut.


55

Telzer e t d . (1975) found that, as shown in Fig. 9, when chromosomes isolated from HeLa cells were attached to grids and incubated with soluble chick brain tubulin before fixation, microtubules assembled only at the kinetochore region, starting in nearly parallel array close to the kinetochore (perpendicular to the long axis of the chromosome) and then radiating outward. No microtubules were visible on isolated chromosomes alone; it seemed likely that the microtubules were initiated at the kinetochore, but the possibility that the elongation of very short stubs of microtubules occurred could not be eliminated.

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ELIZABETH C . RAFF

FIG.9. Microtubule Assembly irr virru on the kinetochore of isolated chromosomes. Chromosomes isolated from HeLa cells were incubated with soluble chick brain tubulin; microtubule assembly took place only at the kinetochore. Marker: I p m . Reprinted from Telzer ef ( I / . (1975). with permission.


57

Finally, as shown in Fig. 10, Gould and Borisy (1977) recently directly demonstrated that the amorphous electron-dense material observed in ultrastructure studies functioned as a microtubule-organizing center in v i m . They studied the formation of microtubules from the centrosomes of CHO cells in culture. This structure consists of a pair of centrioles surrounded by fibrous electron-dense material from which the cytoplasmic microtubules arise in interphase; during mitosis, there is one centrosome at each pole of the mitotic apparatus. These investigators presented electron micrographs of negatively stained material clearly showing that both cytoplasmic and mitotic microtubules arose directly from the amorphous pericentriolar material in normal cells, in cells recovering from colcemid pretreatment, and in lysates of colcernid-blocked cells incubated with soluble pork brain tubulin. Furthermore, microtubules were observed to grow out from isolated preparations of amorphous material (identified as the pericentriolar material by the presence of dense viruslike particles associated with the centrosome in vivo) when incubated with soluble brain tubulin. In the

FIG. 10. The nucleation of microtubule assembly iir rirro by isolated electron-dense pericentriolar material. (a and b) Patches of electron-dense fibrous material isolated from CHO cells in culture; inferred to he pericentriolar material by the presence of viruslike particles typically associated with the material iti v i ~ w~ 4 0 . 8 1 0 .(c and d ) Similar patches of isolated pericentriolar material after incuhation with soluble porcine brain tubulin. ~ 4 0 . 8 1 0 .Reprinted from Could and Broisy (1977). with permission.

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ELIZABETH c‘. RAFF

absence of pericentriolar material, isolated centrioles incubated with brain tubulin showed growth of tubules by direct elongation of centriole tubules. Could and Borisy have concluded that the pericentriolar material in these cells both initiates and anchors microtubules in vivo and in v i m but, as they have also pointed out, their results bring up a very interesting question about the function of the centriole in asters and in cytoplasmic microtubule arrays, and about the origin of microtubule arrays of very different shape and function (i.e., cytoplasmic and mitotic) from the apparently structureless pericentriolar material.

D. EXPERIMENTS IN W H I C H MICROTUBULE ASSEMBLY IS ELICITED in Vivo THE

INJECTION

OF

BY

MICROTUBULE-~R.GANIZING CENTERS I N T O EGGS

Several experiments have been reported in which the formation of asters or mitotic spindles, accompanied by the initiation of cleavage and some degree of embryonic development, is elicited by the injection into eggs of fractions containing microtubules. Such experiments may be considered the in vivo equivalent of the experiments described in the preceding section. Certain observations are common to all these injection experiments. First, at least p a t of the normal temporal sequence of postfertilization events must be preserved; that is, the egg must be capable of being activated, and activation of the egg must occur before cleavage can be initiated. Second, only particulate microtubule-containing structures elicit aster or spindle formation and cleavage initiation. It appears that the injected structures serve as organizing centers for polymerization of the tubulin already present in the egg. As in the in vitro experiments discussed above, microtubule-organizing structures from heterologous species function. Aspects of the results of‘ injection experiments which differ considerably are the type of microtubule-containing structure observed to cause cleavage initiation and the specific nature of the egg response to the injected structures, which varies from formation of multiple asters accompanied by abnormal cleavage to successful completion of parthenogenetic development. It is an old observation in embryology that frog eggs can be induced to undergo parthenogenetic development by pricking with a “dirty” needle. This process consists of two steps: (1) activation of the egg (pricking with a clean needle causes activation without subsequent development), and (2) initiation of cleavage and subsequent development triggered by an agent present in the blood or lymph into which the needle has been dipped. Fraser (1971) examined the extent of parthenogenesis induced in R. pipiens eggs by the injection of various fractions of homogenates of different tissues. Particulate fractions from low-speed centrifugation of homogenates from many tissues from several species elicited cleavage; the highest activity was found in the fraction from brain, which resulted in normal cleavage in 20% of the injected eggs, many of which developed to the hatched tadpole stage. From the effects of colchicine, trypsin, mercap-


59

toethanol, deuterium oxide, and detergents, Fraser concluded that the agent responsible for initiating cleavage was microtubules. She then injected fragments of cilia and flagella axonemes from Tetrahymenu and Chlamydomonas but observed no cleavage. Fraser’s experiments were suggestive but inconclusive because the work was done with uncharacterized fractions. Similar experiments using a fish, the medaka (Oryzias latipes), showed that cleavage could be induced in unfertilized eggs by injecting flagella, purified fragments of sperm tail axonemes, or precipitated aggregates of soluble tubulin, but not by injecting solubilized sperm tail tubulin (lwamatsu and Ohta, 1974; Iwamatsu et al., 1976; Ohta and Iwamatsu, 1974). Heterologous sperm tail fractions elicited cleavage initiation, but the most normal-appearing cleavage resulted from the injection of homologous fractions. In many injected eggs the cleavage patterns were abnormal, resembling polyspermy, but a small percentage developed to gastrulation. Eggs which failed to activate during the injection process failed to cleave at all. The question of timing was recently studied in Ranu eggs by Elinson (1977). He found that cleavage occurred only if both an organizing center was supplied (in this study, by sperm) and activation of the egg had occurred, but that the normal order of these events could be reversed. Oocytes which had been induced to mature by hormone treatment but which were still between metaphase I and metaphase I1 (at which time they become activatable and are normally fertilized) could be inseminated, but no cleavage or development occurred. A small spindlelike structure formed around the sperm chromosomes. Elinson maintained inseminated immature eggs until they became mature and then activated them by electric shock; the eggs then began to cleave and progressed to blastula or blastulalike stages. The cleavage patterns were often irregular, but a small percentage of the eggs completed development to tadpoles. Heidemann and Kirschner (1975) injected basal bodies isolated from Chlumydotnonas and Tetruhymena into unfertilized eggs of X . laevis. They observed the formation of multiple asters and irregular cleavage furrows 20-60 minutes after injection (normally cleavage begins about 90 minutes after fertilization); since both the timing and cleavage pattern were abnormal, the events in the injected eggs probably did not reflect normal events of embryonic development. However, the capacity of the egg to assemble asters depended on the stage of development. Fully grown but immature oocytes were found to contain the same amount of tubulin, measured by colchicine-binding activity, as unfertilized eggs. However, these oocytes could not be fertilized or induced to form asters by the injection of materials which induced aster formation in eggs. Heidemann and Kirschner speculated that tubulin in oocytes is unable to polymerize. More recent work by these investigators has shown that after hormonal stimulation, germinal vesicle breakdown, and mixing of nucleoplasm and cytoplasm have taken place, Xenopus oocytes induced to mature in vitro form asters in response to injected

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ELIZABETH C'. RAFF

basal bodies in the absence of the cortical events of activation (Heidemann and Kirschner, 1978). Aster formation was specific to basal bodies; the injection of soluble tubulin, microtubules prepared from porcine brain, fragments of sperm flagella, buffer alone, or fractions from E. coli had no effect, whereas crude fractions containing centrioles or basal bodies (such as crude nuclei preparations or Tetrahymena pellicle fragments) induced aster formation. No asters formed after the injection of basal bodies plus colchicine. Heidemann and Kirschner suggested that these results support the idea that centrioles play an active role in aster formation, and that whether the basal body or centriole organizes a flagellum or an aster depends on the intracellular environment. Heidemann et a l . (1977) attempted to define some of the components that control the functionality of centrioles in aster formation. They found that aster formation did not occur when basal bodies injected into Xenopus eggs were first treated with proteolytic enzymes or with RNase; treatment with DNase and various other enzymes had no effect. Treatment with proteases was correlated with gross structural damage to the basal bodies, but RNase treatment did not cause any structural damage which they could observe. The effects of RNase appeared to be specific, since treatment of basal bodies with RNase A, a basic protein, or S1 nuclease, an acidic protein, gave the same results. Earlier, however, Dippell (1976) showed that a structure consisting of a twisted fiber and dense granules seen in longitudinal sections of the lumen of the central part of Paramecium basal bodies contained both RNA and protein and that both were removed after either RNase or protease treatment. We have observed similar structures in basal bodies isolated from Chlamydomonas (E. Raff, R. Raff, and F. Turner, unpublished data). Thus it is possible that RNase causes subtle structural changes. However, since Heidemann et al. found that RNase-treated basal bodies served as templates for in vitro microtubule assembly when incubated with soluble brain tubulin, they concluded that the RNA present in basal bodies is required for aster formation and that the two functions of this structure, centriolelike activity (formation of mitotic asters) and basal body-like activity (serving as a direct template for microtubule elongation), are separable. Zackroff et ul. (1976) also investigated the possible functional role of RNA in aster formation using the partly in vitro system of aster formation in homogenates of meiotically dividing Spisula oocytes. However, while RNase treatment in this system also caused changes in aster morphology, the effects were mimicked by other basic proteins such as histones. Contrary to results reported for microtubule assembly in vitro, they observed enhancement of aster formation in the presence of certain polynucleotides and suggested the presence in vivo of an inhibitor of microtubule assembly bound by and thereby inactivated by specific RNA. Maller et al. (1976) reported that the formation of asters and spindlelike structures followed by apparently normal cleavage and development to the blas-


61

tula stage was induced by the injection of sea urchin sperm head fractions into Xenopus eggs. Aster formation and cleavage initiation was correlated with the presence of centrioles in the preparations. Optimal results were obtained with the equivalent of 10 to 15 sperm heads per egg; 90% of injected eggs initiated cleavage at the correct time (90 minutes) and, of these, 16% progressed to blastula stages. Some association of condensed chromosomes with the induced spindles was observed. Thus the events in these eggs resembled normal postfertilization events. With higher doses of injected material, the normal pattern and timing of cleavage was suppressed and precocious multiple cleavage became predominant; after an injected dose of the equivalent of 350 sperm heads per egg, multiple asters formed in all the eggs, followed by rapid irregular fragmentary cleavage such as Heidemann and co-workers had observed. Maller and coworkers suggested that their results indicated a “titration” of the anticleavage threshold of the egg-that the introduction of a gross excess of potential microtubule-initiating sites overwhelms the control system in the eggs. These workers also injected sperm tail fractions, but it required the equivalent of 400 tails per egg to induce parthenogenetic development and only 30% of the eggs initiated cleavage. Because of the large dose needed, they concluded that the source of the activity of tail fractions was likely to be centrioles present from contaminating heads, rather than the flagella microtubules. Forer er nl. (1977) injected mitotic spindles isolated from sea urchin embryos into Rnnu eggs. In half of the eggs injected with glycerol-dimethyl sulfoxide (DMS0)-isolated spindles, or spindles isolated in hexylene glycol and immediately transferred to glycerol-DMSO, normal-appearing cleavage was initiated. The results were the same in enucleated eggs. No cleavage resulted after the injection of spindles isolated and subsequently incubated in hexylene glycol, These investigators preferred the hypothesis that cleavage initiation resulted from function (and chromosome movement) of the injected mitotic figures, but it seems more likely that the injected structures served as microtubule-organizing centers as in other injection experiments. We have examined a maternal effect mutation, nc, in the Mexican axolotl, A . mexicanum (Raff and Raff, 1978; Raff et al., 1976). Sperm enter mutant eggs, which then undergo many of the initial steps of activation such as the cortical response and swelling and migration of the pronucleus, but fail to initiate cleavage or subsequent development. When highly purified fragments of doublet tubules isolated from sea urchin sperm tails were injected into fertilized nc eggs, the eggs initiated cleavage and developed until a blastulalike stage was reached, at which time they arrested. No microtubule assembly or cleavage occurred after the injection of buffer, soluble tubulin, nontubulin particles, or normal nucleoplasm, nor did cleavage take place after the injection of tubule fragments if the eggs were incubated in colchicine. In preliminary experiments we injected basal bodies isolated from Chlamydomonas into activated nc eggs. However, although

62

ELIZABETH C . RAFF

very few of the eggs initiated normal-appearing cleavage, most did not cleave at all and a few underwent multiple premature fragmentary cleavage similar to that routinely observed in Xenopus by Heidemann and Kirschner (1975) and by Maller et ul. (1976) with high levels of injected material. Normal timing was retained in mutant eggs corrected by the injection of tubule fragments. Normal fertile axolotl eggs begin cleavage at 18°C about 7 hours after spawning (and fertilization); fertile nc eggs injected with tubules within 1-2 hours after spawning initiated cleavage 5-6 hours later. Fertilization in axolotl eggs is normally polyspermic; only one sperm nucleus becomes the male pronucleus, but large asters form around each sperm. Sperm enter nc eggs, but no asters form. It is possible that the subsequent initiation of microtubule assembly induced by the injection of relatively large numbers of tubule fragments represents a ‘titration” of the cleavage potential of the egg, as suggested by Maller et al. (1976) for Xenopus. Mutant eggs contain a pool of tubulin essentially similar in size and properties to be found in normal eggs; the lesion in the mutant appears not to reside in the tubulin molecules per se but rather in an activation step preceding the formation or functioning of microtubule-organizing centers. The nc mutant condition could be phenocopied with either normal or nc unfertilized eggs. Unfertilized axolotl eggs can be artificially activated by means of electric shock, and the injection of microtubules into such artificially activated eggs gave the same results as in fertilized nc eggs, that is, initiation of cleavage and development to a partial blastula. In the other injection experiments discussed, the formation of asters was observed by light microscopy. Likewise, examination of sections of fertilized nc eggs or artificially activated axolotl eggs into which microtubule fragments had been injected showed asters and mitotic spindlelike structures in the cleaving blastomeres. As shown in Fig. 11, we also examined these structures by electron microscopy, which revealed that they represented large, parallel arrays of microtubules somewhat similar to those seen in the mitotic spindle of normal cleaving eggs. We occasionally observed doublet microtubules in cross sections of corrected eggs, indicating that the injected doublet tubules retained their morphological integrity. Locating such structures in sections of large, yolky amphibian eggs is technically difficult, and we were not able to examine the site(s) of initiation of the induced microtubules. The various injection experiments confirm that microtubule arrays can be induced to form in vivo by other microtubule-containing structures. The asters formed most likely do not arise through direct elongation of the tubules of the injected basal bodies and flagella axonemes as occurs when such structures are incubated with tubulin in vitro. A possible function of the injected structures might be to serve as a focal point for the amorphous material from which mitotic tubules appear to arise. Similarities in results appear directly related to the general nature of microtubule assembly systems; reasons for differences are less


63

FIG. I I . Microtubules elicited in nr mutant axolotl eggs by the injection of microtubule fragments. Cleavage was induced in fertilized ric eggs by the injection of fragments of purified outer doublet tubules isolated from sea urchin sperm tails; after about 24 hours the resultant blastula-like embryos were fixed and sections were examined by electron microscopy. (a) Longitudinal section of a bundle of microtubules. This array of microtubules is similar to the mitotic microtubules observed in normal embryos. x 15.870. Reprinted from Raff and Raff (1978), with permission. (b) Highermagnification view of another longitudinal section of induced microtubules showing a cross section of an injected doublet tubule (arrow); the inset shows a cross-sectional view of another group of induced microtubules. ~ 5 7 . 2 7 0 .Both electron micrographs were taken by F. R. Turner, Dept. of Biology, Indiana University, Bloomington, Indiana.

clear. The most puzzling difference is that either basal bodies (or centrioles) or microtubule fragments, but not both, elicited aster formation and cleavage initiation in the various species studied. The tempting, if rather unsatisfying, rationalization is to ascribe this to differences in preparative or experimental procedures, or to species differences. For example, of the species studied in the injection experiments, eggs of Runa, the medaka, and Xenopus are all easily activated by pricking, whereas axolotl eggs can be activated only with difficulty by fairly severe electric shock. In other species, notably sea urchins, the formation of asters and even complete parthenogenetic development can be induced without the introduction of microtubule-organizing centers (Brandriff ei al., 1975; Dirksen, 1961; Kato and Sugiyama, 1971; Moy et al., 1977). Microtubule assembly

64

ELIZABETH C:. RAFF

is only one of a complex battery of interlocking events which take place in the egg after fertilization (see Epel, 1977; Epel et af., 1969, 1974); the nature of the coupling between events of activation, microtubule assembly, and cell division may well differ in various species. A N D OTHERSMALL MOLECULES AS POSSIBLE REGULATORS OF E. CALCIUM MICROTUBULE ASSEMBLY in Vivo

Calcium appears to be important in the regulation of microtubule assembly in vivo, although much of the evidence on this point is indirect (reviewed by Bryan,

1975). Of primary consideration is the fact that calcium inhibits microtubule assembly in vitro. Fluctuations in local cellular calcium levels are thus envisioned to participate in microtubule regulation, with relatively high cellular calcium preventing assembly, or alternatively, promoting disassembly, and lower calcium levels allowing assembly. However, how far the in vitro effect can be extrapolated to the conditions in vivo is unclear. As discussed above, the level of calcium which inhibits microtubule assembly in vitro varies considerably, depending on the concentration of magnesium and other ions, but at any rate it has not been reported to be significantly inhibitory at concentrations much below M . According to Rasmussen and Goodman (1975) this is one or even two orders of magnitude greater than the calcium concentrations which exist in mammalian cell cytoplasm. However, as they point out, microsomes and mitochondria have much higher calcium levels (potentially lop2M in mitochondria) although in bound form. Some experiments by Nishida and Sakai (1977) indicated that microtubles may be more calcium-sensitive in vivo than has been observed in vitro. They found that microtubule assembly in crude extracts of porcine brain was 50% inhibited by M calcium, whereas the same extent of inhibition of assembly of microtubule proteins purified by two or more cycles of assembly was achieved at only lo-:$ M calcium. They suggested the presence of a factor controlling calcium effects, since they were able to restore calcium sensitivity to the purified microtubule proteins by adding back approximately equal amounts (measured by protein content) of partially purified supernatant; the relatively large amount of material needed, however, makes this result somewhat difficult to interpret. Circumstantial evidence for calcium control of microtubules is provided by the observations of Gallin and Rosenthal(1974), who found that, during chemotactic movements of human granulocytes, microtubules proliferated in the cell processes concomitantly with changes in the state of cellular calcium levels, including both the release of calcium and a shift of' cytoplasmic calcium to a particulate fraction. Several laboratories have reported experiments aimed at directly assessing calcium effects on microtubules in vivo. Schlaepfer and Bunge (1973) simply


65

exposed axonal microtubules of cultured rat sensory ganglia to the external medium by transecting them; rapid disruption of microtubules occurred in calcium-containing medium but not in the presence of EGTA. Many recent studies of calcium effects have employed the ionophore A23 187 which promotes passive transport of calcium and other divalent cations through lipid barriers, including biological membranes, thus releasing intracellular calcium and bringing it into equilibrium with the surrounding medium (Reed and Lardy, 1972; Wong et d., 1973). Schliwa (1976) studied the effect of the ionophore on the retraction and reformation of heliozoan axopodia; he observed that in the presence of A23 187, disassembly of axopodia microtubules could be controlled by the amount of calcium in the external medium. At lo-” M calcium, slow, orderly disassembly of the axoneme microtubules occurred, and cross sections showed the pattern of remaining microtubules to be intact. At lo-” M calcium, disassembly occurred rapidly, and cross sections showed considerable disarray both in the pattern of remaining microtubules and in individual microtubules (many C and S shapes were present). The axopodia reformed in calcium-free medium containing EGTA. These experiments did not show whether or not calcium is the normal in vivo regulator of axopodiuni length, but Schliwa felt that the levels of calcium which in the presence of ionophore caused retraction of axopodia were low enough to be physiologically significant. Fuller and Brinkley ( I 976) observed that the cytoplasmic microtubule network in cultured 3T3 mouse fibroblasts demonstrated by immunofluorescence staining with antitubulin antibodies disappeared after a 100-minute exposure to medium M calcium. The microtubule network reapcontaining A23187 and 4.9 X peared after the cells were returned to normal medium. As a model for the regulation of assembly by sequestration of calcium, this group added preparations of mitochondria to microtubule assembly mixtures in vitro; mitochondria1 uptake of calcium relieved the inhibition of assembly caused by 5 x M calcium in the presence of equimolar magnesium (Fuller and Brinkley, 1976; Fuller et al., 1975b). Osbom and Weber (1977) tested the effect of calcium on cytoplasmic microtubules directly; they observed that the treatment of cultured mammalian cells with nonionic detergents in buffers containing GTP and EGTA produced a “cytoskeleton” in which the microtubule network remained intact. The inclusion of 4 x M calcium, however, caused disruption and fragmentation of the microtubules. In both these studies the level of calcium observed to cause microtubule disassembly was higher than probable physiological levels. Fulton (1977) observed that, while the transformation of Naegleriu from the ameboid to the flagellated state (during which a cytoplasmic microtubule system develops as well as the flagella) took an hour and required both transcription and translation; the opposite change, back to the ameboid state, required less than a minute. He found that a fraction isolated from amebae caused transient shape changes in flagellates resembling those that took place during actual transforma-

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ELIZABETH C. RAFF

tion. The treatment of flagellates with A23187 caused similar shape changes. Fulton postulated that the microtubule cytoskeleton in the flagellate is rapidly dismantled in the presence of the increased levels of free calcium, which in turn are required for the actin-based motile system in the amebae and that cyclic changes in free calcium levels in vivo are controlled by a factor present in the isolated fraction. Complex changes in both divalent and monovalent ions take place at fertilization and in general in cells changing from a nonproliferating to a mitotic state. Calcium fluctuations and pH changes are involved in the step-up from the quiescent metabolism of the unfertilized egg to initiation of cell division and early embryonic development (reviewed by Epel, 1977). There is some difficulty in sorting out the specific postfertilization events which control assembly of the mitotic apparatus. Treatment with the ionophore A23187 has been found to cause activation of eggs of diverse vertebrate and invertebrate species, apparently through release of intracellular calcium and/or transport of calcium across the cell membrane (Belanger and Schuetz, 1975; Chambers et d . , 1974; Lallier, 1974; Schuetz, 1975; Steinhardt and Epel, 1974; Steinhardt et d.,1974). Ionophore-treated eggs undergo the cortical changes typical of activation and complete meiosis but in most cases do not subsequently begin mitosis. Recent studies with the calciumsensitive photoprotein aequorin have demonstrated that during the activation of both vertebrate and invertebrate eggs a rapid, transient rise in calcium due to the release of intracellular calcium occurs at the surface of the egg; the calcium concentrations reached are on the order of 10-"-10-" M (Gilkey et d.,1978; Ridgway et d.,1977; Steinhardt et [ I / . , 1977). At least in sea urchin eggs, the wave of calcium release is followed by a rise in pH caused by exchange of extracellular sodium ions for intracellular hydrogen ions (Johnson et d.,1976; Lop0 and Vacquier, 1977). Fluctuations in calcium and magnesium ion concentrations appear to regulate the activity of factors which control metaphase arrest and germinal vesicle breakdown in amphibian oocytes (Masui et d . , 1977; Meyerhof and Masui, 1977; Wasserman and Masui, 1976). In other cell types, Jensen et a / . (1977) reported that A23187 had a mitogenic effect on human peripheral lymphocytes, supporting their hypothesis that an increase in the calcium level in some intracellular compartment is an important event in the change to a proliferating state, while Cone and Cone (1976) found that mitosis could be induced in vitro in fully differentiated chick embryo neurons by agents which cause depolarization by increasing intracellular sodium ions and decreasing potassium ions. The complexity of such systems was pointed out by Baltus et d . (l977), who examined ionophore-induced changes in calcium levels in a study of oocyte maturation in Xenopus (which includes formation of a meiotic spindle); these workers concluded that it is an oversimplification to attribute the regulatory role to calcium alone, and that what must be considered is the total balance


67

between mono- and divalent ion levels. Furthermore, they concluded that strong compartmentalization of ions must occur in vivo. Rasmussen and Goodman (1975) also emphasized that changes in calcium levels are accompanied by changes in the total ion balance of the cell. Certainly regulatory mechanisms involving changes in calcium or other ion concentrations must be either temporally transient or spatially localized, or both. For example, while assembly of the mitotic apparatus presumably requires low calcium concentrations, calcium is required for subsequent formation of the cleavage furrow and completion of cytokinesis (Baker and Warner, 1972; Hollinger and Schuetz, 1976; Rebhun, 1977). A calcium-activated ATPase associated with the mitotic apparatus isolated from sea urchin eggs (Mazia et a / . , 1972; Petzelt, 1972; Petzelt and Ledebur-Villiger, 1973) has frequently been postulated to play a role in controlling calcium levels, but there is no direct evidence for this. Implicit in many of the studies mentioned above is the assumption that in analogy to its effects in virro, calcium also acts directly on microtubule assembly in vivo. Another possibility is that it acts indirectly, possibly in coordination with adenosine cyclic 3' 3'-monophosphate (CAMP) or other cyclic nucleotides. Rasmussen and Goodman (1975) have reviewed the relationships between calcium and CAMP, mutually regulatory molecules involved in the translation of cell surface events into metabolic responses within the cell. Many of the known effects of cAMP are mediated through the stimulation of protein kinases with resultant phosphorylation (and a change to the active state) of the target protein(s); in some cases calcium also functions directly in this manner (reviewed by Greengard, 1978). Interest has also focused on a second cyclic nucleotide, cGMP, as a possible microtubule regulator, because of the involvement of guanine nucleotides demonstrated in microtubule assembly in virro and because of the reciprocity of the action of cAMP and cGMP observed in some other cellular processes (see Goldberg er NI., 1974). An interesting possibility exists that some aspect of microtubule assembly may be controlled by cyclic nucleotide-stimulated protein kinase activity through modification not of tubulin but of MAPs. Preferential phosphorylation both of high-molecular-weight MAPs and tau factor by a CAMP-stimulated protein kinase which copurifies with brain tubulin during cycles of polymerization in vitro has been observed in several laboratories (Cleveland et a / . , 1977a,b; Lagnado and Kirazov, 1975; Rappaport er d.,1976; Sheterline, 1977; Sloboda e t a / . 1975, 1976b). Sandoval and Cuatrecasas (1976b) reported that CAMP-stimulated phosphorylation of proteins in brain tubulin preparations was antagonized by cGMP. Unlike calcium, cyclic nucleotides have not been demonstrated to affect microtubule assembly in vitro. However, workers in several laboratories have observed changes in microtubule patterns in various cell types associated with changes in cyclic nucleotide levels (reviewed by Willingham, 1976). The most

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extensive studies have been with CHO cells in culture. When treated with dibutyryl cAMP or other agents which cause elevation of cellular cAMP levels, these cells undergo a marked change in morphology from a knobbed epithelial cell shape to an elongated fibroblast shape; the cell shape change is accompanied by a proliferation of arrays of microtubules in the cell processes, parallel to the cell axis, and appears to be mediated by the stimulation of protein kinase activity (Borman et al., 1975; Hsie et al., 1976; Li et al., 1977; O’Neill et al., 1977; Porteret al., 1974). Rubin and Weiss (1975) determined the amount of assembled microtubules in CHO cells (measured as a percentage of the total colchicinebinding activity pelleted in the presence of a microtubule-stabilizing buffer) and found that the increase in amount of microtubules after dibutyryl cAMP treatment ranged from 30 to 300% depending on culture variables including cell density. Hennebeny et al. (1975) observed that in calcium-containing medium the ionophore A23 I87 prevented the elongation of CHO cells induced by dibutyryl cAMP treatment, suggesting reciprocal effects of calcium and cyclic nucleotides on microtubule assembly in these cells. Microtubule proliferation with concomitant cell elongation has also been observed to accompany increased cellular cAMP levels in several other cell types (Brinkley et al., 1975a,b; Nath et ul., 1978; Willingham and Pastan, 1975). DiPasquale et al. (1976) found that, although the proporition of tubulin in assembled microtubules increased after the treatment of cultured melanoma cells with dibutyryl CAMP, the total amount of tubulin remained unchanged. Several observations, mostly indirect evidence based on colchicine and other drug studies, indicate cGMP-related changes in microtubule patterns in certain cell types. In a study on the capping of binding sites for the lectin concanavalin A (Con A) on peripheral blood polymorphonuclear leukocytes, Oliver (1975) and Oliver et al. (1975) observed that the effects of colchicine and cGMP or agents which stimulated its production were mutually antagonistic, suggesting cGMP promotion of microtubule assembly. Similarly, lysosomal enzyme release in this cell type is inhibited both by colchicine and under conditions where cAMP levels are increased, but is stimulated by cGMP (Goldstein et ul., 1973; Smith and Ignaro, 1975; Weismann et ul., 1975). Kaliner (1977) reported similar observations for immunologic secretion in human lung tissue. Generalizing from the consideration of CHO cells, Puck (1977) has suggested that cAMP mediates the maintenance of the cytoskeleton of microtubules and microfilaments and that this network is involved in the coordination of growthregulating information exchanged between the cell surface and the nucleus; one of the consequences of the disorganization of this network may then be malignant transformation. Observations on CHO and other cells had earlier led to the suggestion that in transformed cells the levels of cAMP are lower than in normal cells; however, not all transformed lines revert to fibroblastlike shape after


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cAMP treatment, nor do all transformed lines have decreased cAMP levels (see reviews by Pastan et al., 1975; Rebhun, 1977). The question of calcium, cyclic nucleotides, and their interrelated involvement in the events of cell division has recently been extensively reviewed by Rebhun (1977), who pointed out that, while coordination of the events of mitosis appears universally to involve changes in the distribution of calcium ions, no generalizations can be drawn concerning the actions of cyclic nucleotides since cell division, and apparently also microtubule assembly, can be stimulated, inhibited, or unaffected by cyclic nucleotides, depending on the cell type in question. Finally, another class of small molecules which may be involved in the regulation of microtubules are sulfydryl-containing compounds; fluctuations in cellular levels of free sulfydryl groups have long been implicated in control of the formation and maintenance of the mitotic apparatus (see Mazia, 1961). In studies on the effects of various metabolic inhibitors on the mitotic apparatus of eggs of sea urchins and other marine invertebrates Rebhun (1976) and Rebhun et al., (1975, 1976) observed that the disassembly of mitotic microtubules was correlated with rises in oxidized forms of glutathione and other sulfhydryl-containing compounds in the cytoplasm; this group suggested that calcium levels might be mediated through the oxidation of sulfhydryls. Mellon and Rebhun (1976a,b) found that, at concentrations of calcium which inhibited polymerization of tubulin in vitro, the titer of free sulfhydryls was also decreased. In studies with human peripheral polymorphonuclear leukocytes, Oliver et al. (1976) and Burchill et ul. (1978) observed the disassembly of cytoplasmic microtubules under conditions in which gluathione oxidation and formation of protein-S-S-glutathione occurred; these workers emphasize that no evidence exists for direct control of microtubules by the interaction of tubulin with glutathione but rather that control of microtubule assembly is part of the general physiological balance of cell metabolism and the cellular response to changes in oxidative conditions. F. TIME-DEPENDENT PROPERTIES OF TUBULIN A N D MICROTUBULES There are numerous reports in the literature that biochemical properties of tubulin may vary depending on the developmental age or stage of differentiation of the tissue from which it is isolated. Such changes in properties may reflect mechanisms which govern the temporal regulation of microtubule assembly and are also part of the general question of tubulin heterogeneity discussed earlier. Some observations are consistent with a single population of tubulin molecules acted upon by changing batteries of regulatory factors, while other observations indicate that different tubulin molecules may be present at different times in development. Both may occur. The timed synthesis of tubulin specific to flagella

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in the ameba-flagellate protozoan Naegleria described by Fulton and co-workers (see discussion above) appears to be a good developmental model system; in higher organisms, gene switches during development are well known. There are recent indications that a general eukaryotic regulatory mechanism may involve the presence of multiple forms of key proteins, perhaps differing only subtly. For example, several of the histones have been found to consist of two or more very closely related proteins present at specific times in development. Relative comparisons of the details of the biochemical properties of tubulin must be made with some care, because the method of preparing tubulin fractions, the method used to measure drug binding, and so on, influence the results (Bamburg etal., 1973a,b; Hains et ul., 1978; Owellen et al., 1972; Raff, 1977; Sherline et al., 1975). Bamburg et al. (1973a,b) first observed that as development progressed the lability of the colchicine-binding activity of tubulin in supernatants from homogenates of chick brain and sensory tissue increased and also that increasing amounts of tubulin occurred in particulate fractions. They suggested that these changes in properties reflected differences in tubulin molecules present at different times during development. Gonzales and Gee1 (1976) observed similar changes in the properties of brain tubulin during the postnatal development of rats. Hains et al. (1978) found that the affinity of tubulin from newborn rat brain for both colchicine and vinblastine differed from that of tubulin from adult rat brain. These workers suggested that in general tubulin in young brain tissue is different from that in adult brain and in light of this offered an intriguing speculation that such differences might explain the fact that the neurotoxicity of vincristine is much less for young children than for adults. The development of brain tissue involves the formation and maintenance of large numbers of neurotubules. Work from two laboratories suggests that at least partial control over this process lies in regulatory factors, possibly the MAPs studied in vitro. Fellous et al. (1975) and Nunez et al. (1975) investigated the extent of microtubule assembly which could be attained in virro in supernatants from homogenates of rat brains of different developmental ages. They found that both the rate of polymerization and the final amount of polymerized microtubules were much lower in preparations from fetal or newborn rats than in those from older rats. The amount of tubulin present, measured by colchicine binding, was about the same and thus did not appear to be the limiting factor in the massive postnatal neurotubule proliferation. Nor was capability for elongation of microtubules the limiting factor; fetal tubulin elongated microtubules when sonicated fragments of adult brain microtubules were added. Polymerization was also stimulated by the addition of adult supernatant (at a concentration which did not support initiation of self-assembly). These workers concluded that an initiation factor(s) was missing in brains at early developmental stages. Fellous et al. (1976) found that MAPs prepared from adult brain as described by Weingarten et


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al. (1975) stimulated the rate of assembly in vitro of brain tubulin from fetal and newborn rats but did not increase the final amount polymerized, which was about half that in supernatants from adult brains. Rat brain maturation is believed to be under thyroid hormone control and in recent work Francon et al. (1977) suggested that thyroxine might be one regulatory signal involved in microtubule assembly. They found that, although brains from hypothyroid and normal rats contained about the same amount of tubulin, the rate of microtubule assembly in virro was much slower in preparations from brains of hypothyroid rats. However, the final extent of polymerization was about the same. The rate of assembly of tubulin from brains of hypothyroid rats was considerably stimulated by the addition of MAPs isolated from normal brain but did not reach the normal rate. However, tubulin made from brains of thyroxine-treated hypothyroid rats assembled at a nearly normal rate. Schmitt er al. (1977) reported similar experiments confirming that tubulin in supernatants from brains of 5-day-old rats polymerized in virro at a much slower rate and to a much lesser extent than tubulin from adult brains. However, in the presence of 4 M glycerol, both the rate and extent of assembly of tubulin from newborn rat brain were similar to those for tubulin from adult brain (which polymerized at the same rate and to the same extent in the presence or absence of glycerol). These workers were unable to correlate the presence of highmolecular-weight MAPs either with the age of the brain or with the rate of in vitro polymerization; they tentatively identified an 82,000-molecular-weight component as being present in tubulin preparations from brains of rats 10 days old or older but not in younger brains. They postulated a membrane-bound initiation factor to explain both the temporal onset and the spatial localization of microtubules in growing neurons. A series of studies related to the question of brain development concern the differentiation of mouse neuroblastoma cells in culture. Schubert et al. (1971) showed that, when these cells were cultured in serum-free medium, they rapidly and reversibly differentiated into a neuronlike morphology, extending long neurites containing numerous microtubules. Protein synthesis was required for the differentiative process. Morgan and Seeds (1975) and Schmitt (1976) showed that the synthesis of tubulin was not a controlling factor, however, since the total tubulin levels remained constant during proliferation of the microtubule network. Recently, Seeds and Maccicmi (1978) suggested that the appearance of microtubules in differentiating neuroblastoma cells might be controlled through MAPs. They examined the ability of high-speed supernatants from cell extracts to promote microtubule assembly in vitro of purified lamb brain tubulin, under conditions in which the brain tubulin did not initiate self-assembly unless brain MAPs were added back. Polymerization was also stimulated by supernatants from differentiated but not from undifferentiated neuroblastoma cells. The stimulation of assembly by differentiated cell supernatant plus brain MAPs was additive; con-

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versely the presence of undifferentiated cell supernatant did not inhibit MAPstimulated assembly. The ability of cell supernatants to stimulate assembly correlated with morphological differentiation; supernatants from cells that were serum-deprived under conditions in which differentiation did not occur, for example, did not stimulate assembly. The differentiated cell supernatants did not contain microtubule fragments nor initiate self-assembly at the concentration used (0.1-0.2 mg/ml); the factor(s) responsible for the brain tubulin assemblystimulating activity was not identified. Mizel and Bamburg (1975) showed that nerve growth factor-induced extension of neurites by chick embryo dorsal root and sympathetic ganglia took place under constant total tubulin levels. Levi et al. (1975) reported that nerve growth factor bound to mouse brain tubulin in vitro and under some conditions stimulated polymerization, but there is no evidence showing that this occurs in vivo. The brain studies involve stages very late in development. Fewer data are available on tubulin properties during early embryonic development. We observed that several changes in the properties of tubulin in supernatants of axolotl egg homogenates, including a marked increase in the lability of colchicinebinding activity, took place after the initiation of cleavage; however, the results of mixing experiments indicated that at least some of the differences in properties were not due to differences in tubulin per se (Raff, 1977). Kuriyama (1977) found no differences in polymerizability in vitro in tubulin from unfertilized and fertilized sea urchin eggs. A question closely related to development and differentiation concerns microtubule regulation during different physiological states. Perhaps the most striking such change is transformation, when both cell morphology and mode of growth alter drastically. As previously discussed, it has been suggested that cytoplasmic microtubule networks are modified during transformation. Biochemical evidence on this point is somwhat unclear. Several groups have compared mouse 3T3 cells with SV40-transformed 3T3 cells; both the polymerization of t u b u h in vitro and the amount of tubulin present were found to be the same in both cell types (Fuller et al., 1975b; Weber et al., 1977a; Wiche et al., 1977b). Fine and Taylor (1976), however, reported that, while the amount of soluble tubulin was the same in both cell lines, the transformed cells had only about two-thirds the normal amount of total tubulin because of a decrease in tubulin appearing in particulate fractions. Ostlund and Pastan (1975) reported that virally transformed rat kidney fibroblasts had only half as much tubulin, measured by colchicine binding, as the parent cells. Pipeleers et al. (1977a,b) examined total tubulin and polymerized microtubule levels in several mammalian cell types under different physiological conditions and concluded that the total tubulin level and the degree of polymerization were independently regulated and could be modulated by many factors. For example, during fasting the total tubulin level in rat and mouse liver fell only slightly,


73

whereas the percentage of tubulin in polymerized microtubules was drastically decreased relative to that in liver from normally fed animals. After glucose feeding, the percentage of polymerized microtubules increased, but the total tubulin level remained constant. G. SPATIAL LOCALIZATION OF MICROTUBULES: ASSOCIATION WITH MEMBRANES A good (teleologically speaking) mechanism for cellular control of the localization andor orientation of microtubule arrays would be to anchor either the initiation sites or the tubules themselves onto some other subcellular structure. It appears that many cells do just that; microtubule-organizing centers, microtubules, and tubulin itself have all been observed in intimate association with membranes. There is both morphological and biochemical evidence on this point. First, in many (though by no means all) cases microtubules arise near membranes or approach them closely. Where the initiation site is associated with membrane, this may imply a way of determining the spatial arrangement of the microtubule array, whereas association with membranes of microtubules which originate elsewhere may imply a funtional rather than a regulatory involvement. In many lower organisms with specialized forms of mitosis, the mitotic microtubule-organizing structures are situated directly on the nuclear membrane (reviewed by Hepler and Palevitz, 1974; Kubai, 1975); several examples of this type were discussed in Section I. In some primitive organisms chromosomal attachment at mitosis is directly to the nuclear membrane; Pickett-Heaps (1969, 1975a) postulated that mitosis arose through such chromosome-membrane connections and that the kinetochore and other microtubule-organizing centers were relics or derivatives of primitive membrane specializations. This hypothesis carries with it the idea that, evolutionarily, microtubules first arose for the purpose of cell division (i.e., mitotic before axoneme microtubules, for instance). The evolutionary shift of chromosome movement from membranes to microtubules has been traced by Kubai (1975). Many other observations discussed in Section I also illustrate membrane involvement in the initiation or placement of microtubules, for example, in the location of protozoan organelles and basal bodies at sites on the cell membrane. The location of centrioles within the cytoplasm may also be membrane-related. Bornens (1977) has recently reported electron micrographs showing that preparations of rat liver nuclear envelopes contain numerous centrioles some of which appear to be physically connected to the membranes by dotlike strands of material of rather low electron density. Ultrastructural examination has revealed the close association of cytoplasmic microtubules in both animal and plant cells (Franke, 1971; Schliwa, 1977; H. J. Wilson, 1970). Such associations are particularly prevalent in neural tissue, and clear associations of microtubules with both pre- and postsynaptic membrane

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specializations have been shown (Bird, 1976; Gray, 1975; Westrum and Gray, 1976, 1977). Observing that the electron-dense aggregates from which tubules arose in the cell center of fish melanophores are in close physical association with elements of the internal cell membrane system, Schliwa (1978) postulated that this represented positioning of the microtubule-organizing structures at sites from which calcium levels could be controlled. Sherline et al. (1977) found that microtubules assembled in vitro bound to isolated pituitary secretory granule membranes. In an ultrastructural study of diploid human fibroblasts in culture using highvoltage electron microscopy Wolosewick and Porter (1976) observed a continuous three-dimensional internal cellular lattice in which microtubules, microfilaments, internal cell membranes, and other cell components were interconnected by 3- to 6-nm filaments. Byers and Porter (1977) observed a similar lattice in cultured fish erythropores; the filaments forniing the lattice were absent during pigment aggregation and reformed when the pigment granules dispersed. These investigators postulated that MAPS might be a component of the filaments. Workers at several laboratories have concluded that microtubules are also closely associated with the plasma membrane. Much of the evidence is derived from studies of the binding of Con A and other lectins to cell surfaces (see reviews by Edelman et a l . , 1976; Nicolson, 1976a,b). In many cases Con A binding is accompanied by clustering or capping of the binding sites; concomitant proliferation andor rearrangement of microtubule arrays immediately adjacent to the cell membrane has been observed (Albertini and Anderson, 1977; Albertini and Clark, 1975; Clark and Albertini, 1976; Hoffstein er al., 1976). In other cell types rearrangements of cell surface components occur only after treatment with agents that disrupt microtubules, including colchicine (Berlin et a l . , 1974; Edelman er al., 1973; Oliver, 1975; Oliver et al., 1975; Yahara and Edelman, 1973, 1975) and calcium ionophores in calcium-containing medium (Poste and Nicolson, 1976). Finally, there is considerable evidence that tubulin is a component of membranes from a variety of sources (reviewed by Stephens and Edds, 1976). Tubulin has been shown to be present in synaptic and postsynaptic membrane fractions (Blitz and Fine, 1974; Walters and Matus, 1975); Estridge (1977) found that some of the tubulin in neural membranes may be exposed on the cell surface. Bhattacharyya and Wolff (1975) showed that tubulin with properties similar but not identical to those of soluble tubulin was a bona fide component of membrane fractions from mammalian brain and thyroid. In subsequent work, Bhattacharyya and Wolff (1976b) found that tubulin solubilized from brain membranes could be copolymerized in vitro with soluble tubulin; these investigators speculated that membrane-bound tubulin functioned in nucleating microtubules but had no direct evidence for this. Stephens (1977a) recently demonstrated that tubulin is the main protein component in the membrane fraction from scallop gill cilia but is


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not present in that from sperm tail flagella; furthermore, the ciliary membrane tubulin was glycosylated to a small extent, whereas ciliary axoneme tubulin was not. As Stephens points out, it is not clear what functional role tubulin plays in the ciliary membrane, nor is it clear why the ciliary and flagellar membranes differ. Related to the question of the association of tubulin with membranes is the existence of other particulate forms of tubulin. Weisenberg (1972a, 1973) examined the organizational state of tubulin before and during meiosis in eggs of the surf clam, S. sofidissima. He made homogenates of eggs by gentle methods in a buffer known to stabilize microtubules, separated them into soluble and particulate fractions, and then determined the distribution of tubulin by colchicine binding. As expected, metaphase eggs contained a significant amount of particulate tubulin in low-speed pellets, presumably representing the mitotic apparatus. The more striking result was that interphase eggs contained the same amount of particulate tubulin (about 10-15% of the total). Unlike that from metaphase, the interphase tubulin-containing particle was very fragile. In the low-speed pellet from gently handled homogenates, Weisenberg isolated granular spheres 10-20 p n in diameter with a membranous structure on one side. The structures contained no microtubules, but microtubules were frequently observed radiating from them. Rougher handling caused this structure to disperse, and the particulate tubulin then appeared in higher-speed pellets. During prophase the amount of tubulin in the particulate fractions declined to a minimum; Weisenberg therefore postulated that the interphase structure was broken down and the component tubulin utilized in assembly of the mitotic apparatus. Weisenberg and Rosenfeld (1975a) speculated that possible functions of the interphase structure could relate to determination of the correct spatial orientation of the mitotic apparatus or to sequestering the specific pool of tubulin from which it would be elaborated. The interphase structure is reminiscent of the basal body and mitotic precursor structures observed in the electron microscope studies discussed in Section I. In fact, Staprans and Dirksen (1974) performed similar experiments to determine the organizational state of tubulin during ciliagenesis in mouse oviduct, which Dirksen (1971) had previously followed in ultrastructure studies (see above). The appearance of increasing amounts of tubulin in the particulate fractions of homogenized newborn mouse oviducts correlated in timing with the presence of the centriole precursors seen in ultrastructure studies. Dirksen and Staprans (1975) found that ciliagenesis was accompanied by a burst of de novo tubulin synthesis and observed by electron microscope autoradiography that at this stage of development more label was present in centriole precursors and basal bodies than in other organelles. However, in a detailed study of labeling patterns during this period of rapid tubulin synthesis Dirksen and Staprans (1977) concluded that the labeling kinetics were too complex to fit a simple model of sequential transfer of tubulin to centriole precursors and then on to the axoneme.

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H. MICROTUBULE REGULATION: GENETIC STUDIES Genetic analysis is a potentially powerful tool for the study of microtubule regulation which has been relatively little exploited. The maternal effect mutation nc in the Mexican axolotl discussed above is an example of the kind of mutation which may help to delineate the points of control over microtubule assembly. An ideal system for examining the developmental control over tubulin and microtubule function is spermiogenesis in Drosophila. Many mutations affecting spermiogenesis have been described, among which are several resulting in altered microtubule assembly or function including disruption of the normal organization of the axoneme (reviewed by Kiefer, 1973; Romrell, 1975). Wilkinson er al. (1974) described a male sterile mutant of D . rnelanogasfer in which the sheath of cytoplasmic microtubules normally attached to the nuclear envelope of developing sperm cells failed to be assembled, with concomitant failure of nuclear shape change; nevertheless, axonemal microtubules assembled. The mutant could be phenocopied by treating wild-type flies with colcemid. Studies of this mutant combined with observations of the effect on spermiogenesis of vinblastine, which prevents assembly of the axoneme but to some extent allows elaboration of the cross-linked nuclear sheath microtubules (Wilkinson et al., 1975), led this group to conclude that the two classes of microtubules necessary for sperm differentiation are separately regulated. Lifschytz and Harevan (1977) and Lifschytz and Meyer (1977) described a series of male sterile sperm dysfunction mutants of D . rnelanogaster including several with defects in the morphology of the meiotic spindle. One of these produced a monastral but dipolar spindle at the second meiotic division, demonstrating independence of the spindle pole from the astral array; two centrioles were present at the single astral pole. These workers concluded that the defects in spindle morphologies observed more likely resulted from defects in the control of microtubule assembly rather than lesions in the structural tubulin gene. The abnormalities observed were confined to meiosis, indicating independent control over other microtubule assembly events. Finally, Rungger-Brandle (1977a,b) reported a mutant of Drosophila hydei in which spermatid differentiation was blocked at an early elongation step. As shown in Fig. 12, electron micrographs of mutant testes cultured in vivo show several microtubule-related abnormalities in morphology which, taken together with the similar changes caused by antimitotic drugs in developing spermatids from normal flies, suggest that the lesion is in microtubule assembly. For example, nuclear and cell elongation failed to take place, apparently because of failure to assemble normal cytoplasmic microtubule arrays; cytoplasmic microtubules were fewer in number than normal, and the majority observed had grossly abnormal cross sections. In addition, the central pair was missing from the flagella axoneme. Interestingly, some of the abnormal microtubule cross sections ob-


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FIG. 12 Abnonnal cytoplasmic microtubules in spermatids of the mutant f(3)pl (lethal polyploid) of L ) . hytlri. Cross sections o f sperinatid microtuhule arrays showing the abundance of various ahnornial forms. The arrows in (d) denote tuhules connected by long linkers. (a) X61.600; (h) ~ 6 1 , 6 0 0(;c ) ~ 8 4 , 7 0 0 ;( d ) ~ 8 3 , 1 6 0 .Reprinted from Rungger-Brindle (1977b). with permission.

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FIG.13 Cytoplasmic microtubules in variant lines of CHO cells in culture after treatment with dibutyryl CAMP. (a) Epithelium-like CHO cell variant showing randomly oriented cytoplasmic microtubules. These cells (with or without dibutyryl cAMP treatment) are similar to untreated normal . Fibroblastlike CHO cells. x 12.325. Inset: Phase-contrast micrograph of a whole cell. ~ 6 8 0 (b) CHO cell variant, showing a long, parallel array of microtubules in the elongated cell processes. Normal CHO cells respond similarly to dibutyryl cAMP treatment, but the fibroblastlike variant cells become even more elongated. ~ 2 8 , 4 7 5 Inset: . Phase-contrast micrograph of a whole cell. ~ 6 8 0 . Reprinted from Borman er a / . (1975), with permission.


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served by Rungger-Brandle in the mutant spermatids resembled those seen by Burton and Himes (1978) after the assembly of brain tubulin in v i m at slightly suboptimal pH levels. Chlaniydomonas is another organism in which mutations involving defects in microtubule assembly have been observed, particularly in formation of the flagella axoneme. Nonmotile mutants with elongated or stumpy flagella or axonemes lacking one or both central pair microtubules or with other defects in microtubule arrangement have all been reported (McVittie, 1972; Pipemo et al., 1977; Warr, 1974; Witman e t a / . , 1978). Goodenough and St. Clair (1975) have described a particularly interesting Chlamydomonas mutant in the basal bodies of which a ring of singlet microtubules is formed instead of the typical triplet tubules. This strain lacks flagella, but occasionally the flagellar transition region, a short axoneme, and the tunnel in the cell wall through which flagella usually emerge do form; the axoneme formed, however, is defective in the same sense as the basal body; that is, it contains only singlet tubules. Cytoplasmic microtubules in this mutant are normal. Control over the number of flagella was illustrated in studies by Warr (1968) of a mutant with a defect in cell division such that multinucleate cells formed; the number of pairs of flagella was the same as the number of nuclei. In addition to mutations there are numerous examples of species in which the axonemal arrangements of the sperm flagella deviate from the typical 9 + 2

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pattern but in which the morphology of the microtubules is normal (for example, see Baccetti et al., 1973, 1974; Phillips, 1974; Schrevel and Besse, 1975; Thomas, 1975; Tulloch and Hershenov, 1967; Van Deurs, 1974). There are also examples in which the microtubules themselves are of variant morphology. Turner (1972) described the ultrastructure of the testis of the water strider, Gerris remigis; the cytoplasm of the sheath surrounding the spermatid bundles is filled with modified microtubules resembling a normal microtubule of typical circular cross section and diameter with one to three curved or hooklike projections. The projections gave some of the modified microtubules the appearance of opened doublet or triplet tubules. Some of the cross sections resembled those discussed above in the D. hydei mutant and in brain tubulin assembled at suboptimal pH. Longitudinal sections showed that the projections were continuous with the tubule wall. The projections disappeared alfter colchicine treatment, leaving microtubules of normal cross section. Baccetti and Dallai (1978) have recently described the feebly motile multiflagellated sperm of the termite Mastotermes durwiniensis in which the centrioles have doublet instead of triplet tubules; also, the flagella axoneme is of an atypical 9 + 0 pattern, and the doublets have only single arms. Two studies have demonstrated abnormal microtubule regulation in mammalian cells in culture. As shown in Fig. 13, Borman et al. (1975) reported a variant line of CHO cells which maintained an epithelium-like shape even after cAMP levels were increased; microtubules appeared in response to cAMP but were randomly distributed and did not give rise to the marked shape changes seen in normal cells; these workers also reported a fibroblastlike variant which normally maintained extensive microtubule arrays. Oliver (1975) and Oliver et a / . (1975) found that some of the deficiencies observed in cells from mutant beige or Chediak-Higashi mice were consistent with abnormalities of microtubule regulation; whereas leukocytes from normal mice required colchicine treatment in order for Con A capping to occur, leukocytes from the mutant mice capped spontaneously. Dooker and Bennett (1974) reported that, in sterile male mice homozygous for the tit" allele marked morphological abnormalities occurred during spermiogenesis, apparently caused by premature disassembly or disorganization of cytoplasmic microtubules. These investigators suggested that disruption of the microtubule arrays was in turn related to abnormalities in the membranes with which these arrays were normally associated. Finally, there have been several reports of various cell types resistant to antimitotic drugs, particularly colchicine. However, there has to date been no demonstration of the existence of the putative modified tubulins. For example, Ling and Thompson (1974) demonstrated that a line of CHO cells resistant to colchicine and vinblastine were less permeable to these drugs than normal CHO cells; mechanisms unrelated to tubulin such as reduced permeability to the drugs,


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increased drug efflux, and the like, have not been unequivocally eliminated in other such studies. 111. The Suppositions: Conclusions about the Control of Microtubule Assembly in Vivo At this point a summary of what is known about the control of microtubule assembly in vivo very nearly constitutes a restatement of the entire problem, but a restatement of the problem may be helpful in assimilating the mass of extraordinarily diverse observations on the topic. Microtubule assembly depends on the formation or activation of microtubule-organizing centers or structures, the primary cellular signals for which are largely unknown. A variety of microtubuleorganizing centers has been observed; the most ubiquitous consists of amorphous electron-dense material, sometimes exhibiting a fibrous or granular substructure, of uncertain biochemical composition. There are some indications that one component of these centers may be tubulin itself, but it is not clear if this material has a general or typical composition. In different cell types or at different times this material may give rise either directly to microtubules or to more elaborate microtubule-containing organelles, particularly centrioles. Centrioles (after some morphological modifications and in some cases after physical relocation to the cell surface) may function as basal bodies and give rise directly to the microtubules of the flagella or cilia axoneme, thus themselves constituting a microtubule-organizing structure. In other cases centrioles may subsequently be associated with the electron-dense material from which either cytoplasmic and mitotic microtubules or other centrioles may arise. Numerous specialized microtubuleorganizing centers of more complex structure exist; most common are spherical or laminated structures composed of electron-dense material resembling that observed in a more amorphous form. The nature of the information which specifies the temporal occurrence and spatial orientation of microtubules is beginning to be defined. Some of the answers are apparent if not explicit; calcium ion fluctuations, membrane involvement, and heterogeneity of tubulin subunits are clearly among the most important regulatory mechanisms. The former two have been appreciated for quite a long time, although the biochemical details are only just now being worked out, while the last point has only relatively recently been understood. One of the most studied current problems concerns the nature and function of nontubulin MAPs. These proteins clearly exist and almost certainly are real and not fortuitously coisolated components of microtubules. The confusing and sometimes contradictory nature of the data concerning their number, identity, and function in vivo may well stem from genuine cellular diversity. There are likely several different kinds of MAPs with perhaps subtly differing and/or interrelated

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functions; MAPS may participate both in the structural scaffolding of the cell a5 well as in the regulation of microtubule assembly or function. A third point which cannot be overemphasized is the difficulty of correlating in vivo observations with in vitro data. The biology of microtubules and the chemistry of tubulin have frequently been considered separate problems, and this may explain apparent anomalies in the two sets of data. A cell may function like a bureaucracy (what is not explicitly permitted is forbidden), while in the more “democratic” in vitro environment of the test tube many tubulin selfassociations are permitted including those which do not occur in the cell. While one may speak in general of the regulation of microtubule assembly, it must be recognized that control must actually occur, perhaps separately, at various levels: that is, control at the cellular level to determine when and where microtubules will be assembled, and control at the molecular level governing the actual process of assembly of tubulin subunits. The latter is the level at which most in vitro data apply. Some regulatory signals may operate exclusively at one level or another. Thus hormonal stimulation of microtubule assembly (such as proliferation of basal bodies in mammalian oviduct in response to estrogen o r neurotubule proliferation in response to thyroxine) clearly are cellular signals removed at least one step from the actual fitting together of microtubule subunits, while guanine nucleotide availability most likely operates at the subunit level. Other signals-such as calcium ions or MAPS-may operate in different ways at both levels. Similarly, there are two aspects of the control over the timing of microtubule appearance: the actual or developmental time at which microtubules must be assembled (or disassembled), and the rate at which tubulin subunits assemble, also an important component of microtubule regulation. Genetic studies may be one of the most helpful approaches in differentiating between different levels of control. It may be a mistake to look for a single mechanism in which all the data can be made to fit into a general statement of how microtubule regulation is achieved. It is at least my own distinct impression that a lot of the data will be left hanging out of the suitcase no matter how hard we sit on it. Just as the obvious long-standing evolutionary constancy of microtubule morphology and tubulin structure for a while obscured the fact of microheterogeneity of tubulin subunits, the large amount of data from studies of assembly of neurotubulin in virro-while of enormous value-may have tended somewhat to obscure the diversity of control mechanisms in other tissues. For example, the mechanisms by which individual microtubules are organized into patterned arrays have clearly been shown to be diverse even in organisms as closely related as various heliozoan protozoa, in some of which an axopodium pattern is established by specific linkage and in others of which it is established by template nucleation. Even the assembly of morphologically indistinguishable organelles-basal bodies and centrioles-has been shown to take place through different morphological steps in different

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organisms (thus the modes of formation in the most extensively studied groups-vertebrates and protists-are quite different and both are radically different from the mode of formation in motile plant sperm). At the conclusion of biochemical reviews it is customary to comment on the necessity for more work on the structure and function of the protein in question; in view of the staggering amount of data already available on tubulin and microtubules perhaps in this case it is more suitable to remark on the need for new analytical insight. Finally, one must remember that control of microtubule assembly is not an isolated problem, but is just one aspect-a central and still puzzling o n e 4 f the general problem of modem biology, initiated in the studies of eighteenth- and nineteenth-century embryologists, of how the morphology of cells and organisms is controlled.

ACKNOWLEDGMENTS I acknowledge with thanks the many people who very kindly sent me the photomicrographs which so elegantly illustrate the topic considered here, R. A. Raff for critical reading of the manuscript, the staff of the Indiana University Department of Biology Office for patient and rapid typing and retyping, and the National Science Foundation for support through Grant PCM 73 02130 A01.

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Control of microtubule assembly-disassembly by calcium-dependent regulator protein.

Role of intermediates in microtubule assembly in vivo and in vitro.

Cell biology. ABG of microtubule assembly.

Magnetic control of protein spatial patterning to direct microtubule self-assembly.

Alterations in microtubule assembly caused by the microtubule-active drug LY195448.

Suppression of microtubule assembly kinetics by the mitotic protein TPX2.

Levuglandin E2 inhibits mitosis and microtubule assembly.

Profilin connects actin assembly with microtubule dynamics.

A protein factor essential for microtubule assembly.

Nuclear assembly shaped by microtubule dynamics.

Inhibition by griseofulvin of microtubule assembly in vitro.

Kinetic analysis of microtubule self-assembly in vitro.

Microtubule motors: many new models off the assembly line.

Ethacrynic acid inhibition of microtubule assembly in vitro.

Nucleotide binding and phosphorylation in microtubule assembly in vitro.

Effects of glutathione-oxidizing agents on microtubule assembly and microtubule-dependent surface properties of human neutrophils.

Guanosinetriphosphatase activity of tubulin associated with microtubule assembly.

The role of divalent cations in the regulation of microtubule assembly. In vivo studies on microtubules of the heliozoan axopodium using the ionophore A23187.

Microtubule stabilization by assembly-promoting microtubule-associated proteins: a repeat performance.

cis-diamminedichloroplatinum (II) (cisplatin) alters microtubule assembly dynamics.

Materials science. How shape affects microtubule and nanoparticle assembly.

Microtubule polymer assembly and transport during axonal elongation.

Tau assembly: the dominant role of PHF6 (VQIVYK) in microtubule binding region repeat R3.

Effects of solution variables on the calcium sensitivity of the microtubule assembly system.