TIBS 17 - AUGUST 1992
EIGHT YEARS and 100 issues of TIBS ago, cell biologists knew a great deal about muscle contraction and ciliary beating, but relatively little about the world of cytoplasmic motility. At that time, it was widely believed that intracellular movements of chromosomes and membrane organelles occurred along microtubules or actin filaments. Molecules similar to muscle myosin and axonemal dynein were also reported to exist in a variety of cells, and hence were suspected to be the agents responsible for cytoplasmic actin- and microtubule-based motility. Although recent work has substantiated this speculation, cytoplasmic myosin and dynein have turned out to be just two of a tremendous number of cytoskeletal-based motors. By 1992, extensive superfamilies of myosins, kinesins and dyneins have been uncovered ]-3, and new motors are being found at a staggering rate. The importance of microtubule-based motility is also becoming better appreciated. In addition to their wellestablished function in flagellar beating, microtubule motors are now known to be involved in protein sorting, the organization of intracellular membranes, the establishment of cell polarity, spindle pole-body separation and chromosome segregation during mitosis and a number of developmental processes. The purpose of this short article is to examine how the discovery of new motors has reshaped our views of cytoplasmic motility. Brief descriptions of the properties of kinesin, dynein and dynamin are presented in the boxes to provide necessary background information, but more detailed information on these microtubule-based motors can be found in a number of comprehensive reviews 4-7.
A populationexplosionof motor proteins Myosin and axonemal dynein were the first motors to be purified, largely because of their great abundance in skeletal muscle and cilia respectively. The highly ordered way in which these motors are arranged also greatly aided efforts to understand the structural
R. D. Vale is at the Departmentof Pharmacology, Universityof California, San Francisco,CA94143, USA.
30O
Microtubule motors: many new models off the assembly line
A far greater variety of microtubule-based motors populate the interior of most eukaryotic cells than was ever imagined, and the inventory of these proteins is growing each year. The discovery of new motors, however, has raised many questions of how cells use their arsenal of force-generating machines. The ability to apply genetics, bacterial expression, biochemistry and in vitro motility assays to study motor proteins provides new opportunities for examining these problems at a molecular level.
basis of motility. In contrast, most cyto- protein of previously unknown funcplasmic motors are present in low con- tion, was shown to be the long soughtcentrations and are not organized in after retrograde motor, cytoplasmic well-defined arrays that can be easily dynein. Dynamin, a GTP-hydrolysing studied by electron microscopy. Hence, protein, was also identified as a micronew approaches had to be developed to tubule motor by such methods, identify motors that might be involved although additional work is required to in processes such as organelle trans- establish that the motility was not proport or mitosis. duced by a kinesin contaminant. The ability to assay the force-genWhile in vitro motility assays and bioerating activities of cytoplasmic motors chemistry overcame the initial barrier in vitro constituted an important break- of finding new motors, molecular genthrough that led to the discovery of etics was responsible for opening a new force-generating molecules. These Pandora's box of new force-generating assays exploited video microscopy8,9, a proteins 1-3. Shortly after its purification, technique that can visualize objects as the kinesin heavy chain was cloned by small as 25 nm microtubules and 40 nm screening a Drosophila cDNA library membrane vesicles in their native state with a polyclonal antibody. In the suband without damaging their function. sequent three years after the sequence With the remarkable clarity provided was published, it was found that muby this approach, it became evident tations causing defects in yeast mating, that the cell interior is bubbling with fungi mitosis, fly meiosis and worm intracellular movements, which myopic axonal transport all reside in genes scientists in the past could never see. encoding ~roteins similar to the kinesin During studies of axonal vesicle trans- heavy chain. The region of sequence port in vitro by video microscopy, it homology was confined to the aminowas discovered serendipitously that terminal 350--400 amino acid domain of microtubules were propelled across the kinesin, which produces microtubule surface of a glass microscope slide by a movement in vitro when expressed in non-specifically adsorbed protein 1°,1]. Escherichia coli as a fusion protein 12. This microtubule translocation assay, Thus, the ldnesin-related proteins, by in conjunction with biochemical virtue of their homology to kinesin's fractionation techniques, led to the motor domain, are also thought to funcpurification of kinesin, a novel motor tion as mic.rotubule motors. This supprotein that moves towards the plus position, however, has only been demends of microtubules (defined as the onstrated for ncd from Drosophila [the fast-growing end from polymerization protein encoded by non-claret disjuncstudies). This direction would corre- tional (ncd)] 13,14and Eg5 from Xenopus spond to transport towards the nerve laevis (K. Sawin, K. LeGuellec, terminal in an axon (anterograde trans- M. Phillipe and T. Mitchison, pers. comport). Using a similar in vitro motility mun.), which both induce microtubule assay, MAP1C, a microtubule-binding movement in vitro. © 1992,ElsevierSciencePublishers, (UK) 0376-5067/92/$05.00
TIBS 17 - AUGUST 1992
KJlle~n
Conventional kinesin is a p l u s - e n d - ~
microtubule motor. It is an
(a)
10 nm
20 nm
80 nm
14 n m
(b)
amino acids 400
0 I
I
ATP
Microtubule Motor
800 I
I
(x-Helical coiled-coil Stalk
? Tail
(c) Latex bead
There is no significant amino acid identity between members of the kinesin superfamily in their non-motor or 'tail' domains. The considerable differences in the predicted primary and secondary structures of tails has led to the notion that they bind to and transport different types of 'cargo' in the cell. Although it is likely that this domain targets motors to different sites in the cell via specific receptors, this hypothesis has not yet been substantiated by direct experimental evidence. Kinesin and kinesin-like motor amino acid sequence information obtained by genetic screens gave rise to strategies of finding additional members of the kinesin superfamily. From comparing the various kinesin sequences, an amino acid 'signature' of the kinesin motor domain began to emerge. Based upon regions of high amino acid conservation, oligonucleotide primers for the polymerase chain reaction (PCR) were designed 32,3aand anti-peptide antibodies were produced 34,3s, which were used to identify many new kinesin motors. From such studies, the number of kinesin superfamily members in a single organism appears to be quite large. At least 12 genes encoding kinesin-like proteins have been found in Drosophila s2,
and other evidence suggests that over 18 more may be lurking in the genome 33. Kinesin motors also continue to be uncovered in unexpected ways, as illustrated by the discovery of SMY1 as a kinesin-like motor in Saccharomyces cerevisiae 15. The gene for SMY1 was identified by screening for suppressor mutations of a myosin mutation (myo2) that causes a defect in secretion during budding. Surprisingly, the gene does not encode another myosin motor, but a protein containing a kinesin motor domain. The connection between this putative microtubule motor and actinbased motility is intriguing, but still unresolved. The inventory of dynein motors is also likely to expand in the next few years. The sequence of a dynein heavy chain (13 heavy chain from sea urchin flagella outer arm dynein, 4 466 amino acids long) was recently obtained 16,~7 and did not show homology to kinesin or any other protein. Since dynein does not harbor a kinesin motor domain, these two motor types must have evolved either independently or from a common nucleotidase in the very distant past. PCR is now being used to clone many other dynein heavy chains from a variety of organisms. The se-
quences from these other heavy chains should help to define the extent and signatures of dynein's motor domain. What surprises will be in store for the dynein field? As was true for kinesin, molecular genetics could uncover divergent types of dyneins (minidyneins?) as well as mutations in cytoplasmic dynein genes, which might help to understand their biological functions.
Motor amino acid sequences:what do they tell us? The wealth of sequence information will ultimately be important for under-
standing how motor proteins produce movement, although most of the clues derived from sequence comparisons are enigmatic at the present time. So little is known about the relationship between primary structure and motor function that care must be taken in concluding that an amino acid homology implies a functional similarity. To illustrate this l~oint, it was generally believed that all kinesin-like motors would elicit movement with the same polarity as conventional kinesin, which is a plus-end-directed microtubule motor. Hence, it came as quite a surprise when ncd expressed from E. coli was found to
301
TIBS 17 - AUGUST 1992
Dynein Dyneins are micmtuDule-stimulated ATPases that induce movement towards the minus =ends of microtubules. It [(b)] is characterized by an enormous ~500 kD~apolypeptide that contains several consensus sites for nucteotide binding; only one of the sites seems clearly to be involved in ATP hydrolysis. Dyneins from different sources contain either one, two or three distinct heavy chains and appear correspondingly as one-, two- or three-headed molecules by electron microscopy [flagellar outer arm dynein is depicted here (a)]. These motors also contain several types of associated polypeptideS (14-120 kDa); the positions of all the light chains are not known, although at least some appear to be located near the base of the molecule. The reason fOr dynein's enormous size and-complex polypeptide cOmposition is unknown, altl-~ugh much of this protein is thought to perform regulatory functions as well as anchor the dynein to appropriate target sites. Several forms of dynein comprise the outer and inner arms of flagellar and ciliary axonemes, and each is thought to have a distinct role in the beating of flagella and cilia. A cytoplasmic form of dynein is thought to
I4!
(a)
13.4 nm
nm
(b)
amino acids 2 000
0 I
I
!
NH21
4 000 [
I
II ATP~' ~ ~' t l Nucleotide-binding motifs
t-COOH
t Coiled-coils?
erated during ciliary and flagellar beating~
move towards the microtubule minusend 13,~4.From recent experiments where the kinesin tail domain was joined to ncd, it was shown that the type of motor domain, and not the tail, determines the direction of movement (R. Stewart and L. Goldstein, pers. commun.). Since the motor domains of kinesin and ncd are very similar to one another (40% amino acid identity), apparently subtle structural changes can have such profound consequences on motor function. It might therefore even be presumptuous to conclude, on sequence information alone, that SMY1 from S. cerevisiae is a microtubulebased and not an actin-based motor. Perhaps in the future, the structural determinants of directionality and other parameters of motor function will be understood, but for now, the functions of motors cannot be inferred simply from their DNA sequences. While sequence information alone does not tell us how motors work, it is valuable for directing new biochemical experiments. As an example, the dynein heavy chain sequence reveals the presence of at least four consensus sites (phosphate binding loops) for nucleotide binding, which was somewhat surprising, since all other known motors (myosin, kinesin, helicases) contain only a single site for ATP binding. One of the dynein sites appears to be where nucleotide hydrolysis takes place, but the functions of the other three sites are unclear. Since these motifs are conserved among several dynein isoforms m sea urchin (I. Gibbons, pers. commun.), it seems likely that the multiple sites serve some important role. Is dynein a four-cylinder motor, or do some
302
of the ATP sites serve an allosteric function? Biochemical investigations will ultimately be required to sort out this problem (perhaps in combination with in vitro mutagenesis), but the sequence information derived from cDNA cloning is responsible for bringing this intriguing question to the fore. Amino acid sequences of the tail domains of kinesin-like proteins have unfortunately been less revealing than those from the 'head' (motor) domain. Although the tail domain of conventional kinesin is highly conserved between human, fly, squid and sea urchin, none of the tail domains of the kinesin-like proteins show any sequence similarity to one another, or to any other known protein sequence. This is somewhat surprising, since some of the kinesinlike proteins seem to be involved in similar functions. For example, CIN8'8 (S. cerevisiae), KIPP 9 (S. cerevisiae), bimC 2° (Aspergillus nidulans), and cut72~ (Schizosaccharomyces pombe) all appear to participate in spindle pole-body separation at the onset of mitosis. An evolutionary relationship between these proteins is suggested by the fact that their motor domains are more highly conserved to one another (~60% amino acid identity) than to other kinesins (30-40% identity). These findings suggest that the tail domains of kinesin motors can diverge considerably during the course of evolution, yet still carry out the same function. It is also possible, however, that the exact homologues of the above mentioned kinesin-like proteins have still not been identified and sequenced from these different species. In addition to providing sequence information, cDNA clones are proving
to be powerful tools for studying the biochemistry of motors. Cytoplasmic motor proteins are present in very low abundance in most cells, which imposes a serious impediment to X-ray crystallography, pre-steady state kinetics determinations and other studies requiring large quantities of pure protein. However, the ability to express active motors in E. coli 12q4 now makes biochemical and functional analysis of any motor a feasible undertaking. Different domains of a motor protein can also be expressed separately for functional or crystallization studies. For example, expressed motor domains can be used to study chemomechanical transduction, and expressed tail domains should be useful reagents for identifying motor receptors. However, since most forcegenerating enzymes contain associated light chains, studying expressed heavy chains alone will not provide a complete understanding of how motors function and are regulated in vivo. Thus, motors also must be biochemically purified in their native state in order to determine their quaternary structure and to study their functional properties.
Motor superfamilies - why so many models to choose from? Deciphering the biological roles of the numerous motors being discovered is an important, although somewhat daunting endeavor. One could imagine that each motor is highly specialized for a unique force-generating task. By such reasoning, the extent of motor superfamilies would reflect the diversity of microtubule force-generating activities that the cell has to perform.
TIBS 1 7 - A U G U S T 1 9 9 2
PylOn Dynamin is a microtubule-activatedGTPaseT M that belongs to a superfamily of proteins sharing homology in the amino-terminalGTP-binding domain3°. Although initially reported to function as a microtul0ule motor, recently it has become uncertain whether this protein possesses force-generating capability. Further work is needed to settle this issue. Dynaminconsists of a single I 0 0 kDa polypeptidethat contains a -300 amino acid amino-terminal globular domain with a consensus GTP-binding site and a carboxy-terminaldo.main that binds to microtubules (see Rg.; J. Herskovitz and R. Vallee, pets. commun.). Dynamin mutations in Drosophila (shibire) produce defects in the endocytosis and recycling of synaptic vesicles at nerve terminals. The rela-
0 i
!
NH2"I
amino acids 400 ! ~
~
C
800 i
i O
O
H
Microtubule-binding region
GTP
Region of homology with Mx, VPS1/SPO15
I~S~ and an E. coil protein i n v o ~ in chromoso~ partitioning (MokB). The common f u s i o n linking these proteins ,et been ascertained, but isan active area of investigation. . . .
Clearly, there is some truth to this idea; however, strictly speaking, a paradigm of 'one task - one motor' is inconsistent with recent data. For example, yeast mutants defective in either of two kinesin-like proteins (KIP1 and CIN8) are viable and have little or no abnormal phenotype; however, the double mutant is not viable due to a defect in the assembly of the mitotic spindle ~8J9. Such findings suggest an overlapping and redundant function of these kinesin-like motors. Individual motors can also perform multiple tasks. For example, mutational analyses show that KAR3, a kinesin-like protein in S. cerevisiae, produces nuclear migration during mating and also performs some as yet undefined function in mitosis. Another example of a multifunctional motor is cytoplasmic dynein, which in mammalian cells has been implicated as a motor for membrane organelles in interphase and chromosome movements during mitosis. The apparent divergence of cytoplasmic motors in various species may also reflect the multitude of ways in which organisms carry out force-generating tasks. For example, the strategies for segregating chromosomes during mitosis and meoisis can vary considerably. In mammalian cells, separation is accomplished primarily by chromosomes moving along kinetochore fibers towards the pole, while in fungi, separation is accomplished primarily by pushing the two spindle poles apart from one another. Some force-generating processes also appear to be rather unique to particular organisms. Examples include microtubule-based delivery of nurse-cell contents to the oocyte during insect embryogenesis or the microtubule-dependent exchange of micronuclei during Tetrahymena conjugation. Thus, although tubulin and
motor domains are highly conserved between species, it seems as though objects can be pushed and pulled in cells in a variety of different ways, which produce equally effective outcomes. Customizing the engine
The force-generating properties of motors also appear to be well adapted to suit the biological context in which they operate. For instance, the transport of membrane vesicles appears to be driven by very few motors, in some instances perhaps by only one. To meet this challenge, kinesin (and probably cytoplasmic dynein) remains bound to microtubules for the vast majority (>99%) of its ATPase cycle22.23;this prolonged attachment time enables single motor molecules to transport organelles for long distances without falling off the microtubule. In contrast, thousands of dynein molecules interact with adjacent outer doublet microtubules in the axoneme, which makes it unnecessary for individual motors to remain persistently and strongly bound to microtubules. Indeed, in vitro motility data (R. Vale, unpublished) seem to confirm that axonemal dynein is strongly microtubule-bound for only a short portion of the hydrolysis cycle. Some motors are also built for speed, while others produce slow and steady movement. The fast beating of cilia and flagella requires that axonemal dyneins produce rapid microtubule sliding (>10 lim sec-~); chromosome motors, on the other hand, operate at 100-1 000-fold slower rates. Some properties of microtubulebased motors are also so remarkable that they seem to challenge conventional ideas of how chemomechanicai enzymes work. Certain inner arm axonemal dyneins, for example, rotate microtubules about their longitudinal
axis as they move them forward across a glass surface, indicating that they produce torque as well as a forward propulsion 24. Such movement is difficult to explain without evoking some modification of the traditional swinging crossbridge model, which envisages that the motor changes its angle relative to the filament and produces movement by an oar-like mechanism. Even more surprising and difficult to explain mechanistically is the finding that kinesin and the kinesin-like motor ncd move in opposite directions along the microtubule ~3,14. Perhaps, by understanding what specifies the directon of movement, one may also get at the heart of the chemomechanical transduction mechanism itself.
Outlook Eight years ago, progress in cytoplasmic motility was thwarted by an almost complete lack of information on intracellular protein motors. Today, with a number of motors known and reagents available (e.g. cDNA clones, antibodies), it is relatively straightforward to identify a variety of kinesins (and in the near future dyneins) from almost any eukaryotic organism. The greater challenge now comes in deciphering the logic of how organisms use their multitude of force-generating machines and in understanding how motors produce movement from chemical energy. Although these problems are difficult, the problem of microtubule motors has brought together an excellent group of scientists with diverse backgrounds and expertise. Individuals, who eight years ago were studying worm development, axonal transport or the kinetics of the myosin ATPase, now find themselves studying the same collection of microtubule motors. If information gathering
303
TIBS 17 - AUGUST 1992
continues at the present rate, the world of cytoplasmic motility will be very different again by th$ time the 300th issue of TIBS is published.
References 1 Endow, S. A. ($991) Trends Biochem. Sci. 16, 221-225 2 Goldstein, L. S. B. (1991) Trends Cell Biol. 1, 93-98 3 Vale, R. D. and Goldstein, L. S. B. ($990) Cell 60, 883-885 4 Bloom, G. S. (1992) Curr. Op. Cell Biol. 4, 66-73 5 Porter, M E. and Johnson, K. A. (1989) Annu. Rev. Cell Biol. 5, 119-151 6 Schroer, T. A. and Sheetz, M. P. (1991) Annu. Rev. Physiol. 53, 629-652 7 Vallee, R. B. and Shpetner, H. S. (1990) Annu. Rev. Biochem. 59, 909-932 8 Allen, R. D. (1985) Annu. Rev. Biophys. Biophys. Chem. 14, 265-284 9 Inoue, S. (1986) Video Microscopy, Plenum 10 Allen, R. D. et al. (1985) J. Cell Biol. 100, 1736-1752
11 Vale, R. D., Schnapp, B. J., Reese, T. S. and Sheetz, M. P. (1985) Cell 40, 559-569 12 Yang, J. T. et al. (1990) Science 249, 42-47 13 McDonald, H. B., Stewart, R. J. and Goldstein, L. S. (1990) Cell 63, 1159-1165 14 Walker, R. A., Salmon, E. D. and Endow, S. A. (1990) Nature 347, 780-782 15 Lillie, S. H. and Brown, S. S. (1992) Nature 356, 358-361 16 Gibbons, I. R., Gibbons, B. H., Mocz, G. and Asai, D. J. (1991) Nature 352, 640-643 17 Ogawa, K. (1991) Nature 352, 6 4 3 4 4 5 18 Hoyt, M. A., He, L., Loo, K. K. and Saunders, W. S. J. Cell Biol. (in press) 19 Roof, D. M., Meluh, P. B. and Rose, M. D. J. Cell Biol. (in press) 20 Enos, A. P. and Morris, N. R. (1990) Cell 60, 1019-1027 21 Hagan, I. and Yanagida, M. (1990) Nature 347, 563-566 22 Block, S. M., Goldstein, L. S. and Schnapp, B. J. (1990) Nature 348, 348-352 23 Howard, J., Hudspeth, A. J. and Vale, R. D. (1989) Nature 342, 154-158 24 Vale, R. D. and Toyoshima, Y. Y. ($988) Cell 52,
459-469 25 Ferreira, A. et al. (1992) J. Cell Biol. 117, 595-606 26 Hollenbeck, P. J. and Swanson, J. A. (1990) Nature 346, 864-866 27 Pfister, K. K. et aL ($989) J. Cell Biol. 108, 1453-1464 28 Saxton, W. M., Hicks, J., Goldstein, L. S. B. and Raft, E. C. (1991) Cell 64, 1093-1102 29 Hackney, D. D., Levitt, J. D. and Suhan, J. (1992) J. Biol. Chem. 267, 8696-8701 30 Obar, R. A., Collins, C. A., Hammarback, J. A., Shpetner, H. S. and Vallee, R. B. (1990) Nature 347,256-261 31 Shpetner, H. S. and Vallee, R. B. (1992) Nature 355, 733-735 32 Stewart, R. J., Pesavento, P. A., Woerpel, D. W. and Goldstein, L. S. B. (1991) Pro& Natl Acad. Sci. USA 88, 8470-8474 33 Endow, S. A. and Hatsumi, M. (1991) Proc. Natl Acad. Sci. USA 88, 4424-4427 34 Cole, D. G. et al. (1992) J. Cell Sci. 101, 291-301 35 Sawin, K. E., Mitchison, T. J. and Wordeman, L. G. ($992) J. Cell Sci. 101, 303-313
OBITUARY Peter Mitchell 1920-1992
the first clear statement of the electroOnly rarely do individual scientists leave chemical proton gradient as the an indelible mark on a field. Peter coupling intermediate, the need for a Mitchell made such a mark when he membrane with low proton permeability, proposed and then developed and and the mechanism by which dinitrodefended the chemiosmotic theory of phenol uncouples oxidative phosphorylenergy coupling in oxidative phosation. None of these proposals was supphorylation, photophosphorylation and ported by experimental evidence at the active transport systems. time, as illustrated by the fact that the Mitchell obtained his PhD in biooriginal hypothesis had the direction of chemistry from Cambridge, with J. F. Danielli, and particularly appreciated proton movements backwards. his contact there with David Keilin. He Experimental work by Mitchell and Jennifer Moyle soon provided the evilater moved to the University of Edindence that mitochondria do transport burgh, and, in 1963, moved to a ruined protons in the manner proposed, but it great house near Bodmin, Cornwall. took about ten years to convince the Mitchell designed the restoration of majority of biochemists that the theory Glynn House to contain his family apartments and research laboratories, and was fundamentally correct. Mitchell's worked on the project for two years characterization of this evolution in scientific thinking was typically gracious: alongside the craftsmen. Mitchell and 'most remarkable and admirable is the his brother then endowed the Glynn altruism and generosity with which the Research Foundation with £240000 of their inheritance, creating an institution former opponents of the "chemiosmotic which is still very active. hypothesis" not only came to accept it, In part, his work reflected a conbut actively promoted it to the status of scious effort from his student days to membranes by electron transfer as an a theory'2. bridge a gap between scientific fields. integral part of respiration. Mitchell We can safely say that Mitchell's Chemical reactions belonged to chem- extended this idea to include a proton work revolutionized biochemistry and istry, electric fields belonged to phys- transporting ATPase, thus explaining transport physiology, but Mitchell did ics, and processes containing elements ATP synthesis in mitochondria and not believe that science was advanced of both were likely to be ignored. The chloroplasts. But the brilliance of his by revolution: 'let me hasten to remark plant physiologists E. J. Lund and H. proposal came from the completeness ... that I make no claim to biochemical Lundegardh had proposed that electric of its construction. His paper in Nature ~ originality, except, perhaps, inasmuch fields are generated across biological not only proposed the ATPase, but was as I have consciously endeavored to © 1992,ElsevierSciencePublishers,(UK) 0376-5067/92/$05.00 304
EIGHT YEARS and 100 issues of TIBS ago, cell biologists knew a great deal about muscle contraction and ciliary beating, but relatively little about the world of cytoplasmic motility. At that time, it was widely believed that intracellular movements of chromosomes and membrane organelles occurred along microtubules or actin filaments. Molecules similar to muscle myosin and axonemal dynein were also reported to exist in a variety of cells, and hence were suspected to be the agents responsible for cytoplasmic actin- and microtubule-based motility. Although recent work has substantiated this speculation, cytoplasmic myosin and dynein have turned out to be just two of a tremendous number of cytoskeletal-based motors. By 1992, extensive superfamilies of myosins, kinesins and dyneins have been uncovered ]-3, and new motors are being found at a staggering rate. The importance of microtubule-based motility is also becoming better appreciated. In addition to their wellestablished function in flagellar beating, microtubule motors are now known to be involved in protein sorting, the organization of intracellular membranes, the establishment of cell polarity, spindle pole-body separation and chromosome segregation during mitosis and a number of developmental processes. The purpose of this short article is to examine how the discovery of new motors has reshaped our views of cytoplasmic motility. Brief descriptions of the properties of kinesin, dynein and dynamin are presented in the boxes to provide necessary background information, but more detailed information on these microtubule-based motors can be found in a number of comprehensive reviews 4-7.
A populationexplosionof motor proteins Myosin and axonemal dynein were the first motors to be purified, largely because of their great abundance in skeletal muscle and cilia respectively. The highly ordered way in which these motors are arranged also greatly aided efforts to understand the structural
R. D. Vale is at the Departmentof Pharmacology, Universityof California, San Francisco,CA94143, USA.
30O
Microtubule motors: many new models off the assembly line
A far greater variety of microtubule-based motors populate the interior of most eukaryotic cells than was ever imagined, and the inventory of these proteins is growing each year. The discovery of new motors, however, has raised many questions of how cells use their arsenal of force-generating machines. The ability to apply genetics, bacterial expression, biochemistry and in vitro motility assays to study motor proteins provides new opportunities for examining these problems at a molecular level.
basis of motility. In contrast, most cyto- protein of previously unknown funcplasmic motors are present in low con- tion, was shown to be the long soughtcentrations and are not organized in after retrograde motor, cytoplasmic well-defined arrays that can be easily dynein. Dynamin, a GTP-hydrolysing studied by electron microscopy. Hence, protein, was also identified as a micronew approaches had to be developed to tubule motor by such methods, identify motors that might be involved although additional work is required to in processes such as organelle trans- establish that the motility was not proport or mitosis. duced by a kinesin contaminant. The ability to assay the force-genWhile in vitro motility assays and bioerating activities of cytoplasmic motors chemistry overcame the initial barrier in vitro constituted an important break- of finding new motors, molecular genthrough that led to the discovery of etics was responsible for opening a new force-generating molecules. These Pandora's box of new force-generating assays exploited video microscopy8,9, a proteins 1-3. Shortly after its purification, technique that can visualize objects as the kinesin heavy chain was cloned by small as 25 nm microtubules and 40 nm screening a Drosophila cDNA library membrane vesicles in their native state with a polyclonal antibody. In the suband without damaging their function. sequent three years after the sequence With the remarkable clarity provided was published, it was found that muby this approach, it became evident tations causing defects in yeast mating, that the cell interior is bubbling with fungi mitosis, fly meiosis and worm intracellular movements, which myopic axonal transport all reside in genes scientists in the past could never see. encoding ~roteins similar to the kinesin During studies of axonal vesicle trans- heavy chain. The region of sequence port in vitro by video microscopy, it homology was confined to the aminowas discovered serendipitously that terminal 350--400 amino acid domain of microtubules were propelled across the kinesin, which produces microtubule surface of a glass microscope slide by a movement in vitro when expressed in non-specifically adsorbed protein 1°,1]. Escherichia coli as a fusion protein 12. This microtubule translocation assay, Thus, the ldnesin-related proteins, by in conjunction with biochemical virtue of their homology to kinesin's fractionation techniques, led to the motor domain, are also thought to funcpurification of kinesin, a novel motor tion as mic.rotubule motors. This supprotein that moves towards the plus position, however, has only been demends of microtubules (defined as the onstrated for ncd from Drosophila [the fast-growing end from polymerization protein encoded by non-claret disjuncstudies). This direction would corre- tional (ncd)] 13,14and Eg5 from Xenopus spond to transport towards the nerve laevis (K. Sawin, K. LeGuellec, terminal in an axon (anterograde trans- M. Phillipe and T. Mitchison, pers. comport). Using a similar in vitro motility mun.), which both induce microtubule assay, MAP1C, a microtubule-binding movement in vitro. © 1992,ElsevierSciencePublishers, (UK) 0376-5067/92/$05.00
TIBS 17 - AUGUST 1992
KJlle~n
Conventional kinesin is a p l u s - e n d - ~
microtubule motor. It is an
(a)
10 nm
20 nm
80 nm
14 n m
(b)
amino acids 400
0 I
I
ATP
Microtubule Motor
800 I
I
(x-Helical coiled-coil Stalk
? Tail
(c) Latex bead
There is no significant amino acid identity between members of the kinesin superfamily in their non-motor or 'tail' domains. The considerable differences in the predicted primary and secondary structures of tails has led to the notion that they bind to and transport different types of 'cargo' in the cell. Although it is likely that this domain targets motors to different sites in the cell via specific receptors, this hypothesis has not yet been substantiated by direct experimental evidence. Kinesin and kinesin-like motor amino acid sequence information obtained by genetic screens gave rise to strategies of finding additional members of the kinesin superfamily. From comparing the various kinesin sequences, an amino acid 'signature' of the kinesin motor domain began to emerge. Based upon regions of high amino acid conservation, oligonucleotide primers for the polymerase chain reaction (PCR) were designed 32,3aand anti-peptide antibodies were produced 34,3s, which were used to identify many new kinesin motors. From such studies, the number of kinesin superfamily members in a single organism appears to be quite large. At least 12 genes encoding kinesin-like proteins have been found in Drosophila s2,
and other evidence suggests that over 18 more may be lurking in the genome 33. Kinesin motors also continue to be uncovered in unexpected ways, as illustrated by the discovery of SMY1 as a kinesin-like motor in Saccharomyces cerevisiae 15. The gene for SMY1 was identified by screening for suppressor mutations of a myosin mutation (myo2) that causes a defect in secretion during budding. Surprisingly, the gene does not encode another myosin motor, but a protein containing a kinesin motor domain. The connection between this putative microtubule motor and actinbased motility is intriguing, but still unresolved. The inventory of dynein motors is also likely to expand in the next few years. The sequence of a dynein heavy chain (13 heavy chain from sea urchin flagella outer arm dynein, 4 466 amino acids long) was recently obtained 16,~7 and did not show homology to kinesin or any other protein. Since dynein does not harbor a kinesin motor domain, these two motor types must have evolved either independently or from a common nucleotidase in the very distant past. PCR is now being used to clone many other dynein heavy chains from a variety of organisms. The se-
quences from these other heavy chains should help to define the extent and signatures of dynein's motor domain. What surprises will be in store for the dynein field? As was true for kinesin, molecular genetics could uncover divergent types of dyneins (minidyneins?) as well as mutations in cytoplasmic dynein genes, which might help to understand their biological functions.
Motor amino acid sequences:what do they tell us? The wealth of sequence information will ultimately be important for under-
standing how motor proteins produce movement, although most of the clues derived from sequence comparisons are enigmatic at the present time. So little is known about the relationship between primary structure and motor function that care must be taken in concluding that an amino acid homology implies a functional similarity. To illustrate this l~oint, it was generally believed that all kinesin-like motors would elicit movement with the same polarity as conventional kinesin, which is a plus-end-directed microtubule motor. Hence, it came as quite a surprise when ncd expressed from E. coli was found to
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Dynein Dyneins are micmtuDule-stimulated ATPases that induce movement towards the minus =ends of microtubules. It [(b)] is characterized by an enormous ~500 kD~apolypeptide that contains several consensus sites for nucteotide binding; only one of the sites seems clearly to be involved in ATP hydrolysis. Dyneins from different sources contain either one, two or three distinct heavy chains and appear correspondingly as one-, two- or three-headed molecules by electron microscopy [flagellar outer arm dynein is depicted here (a)]. These motors also contain several types of associated polypeptideS (14-120 kDa); the positions of all the light chains are not known, although at least some appear to be located near the base of the molecule. The reason fOr dynein's enormous size and-complex polypeptide cOmposition is unknown, altl-~ugh much of this protein is thought to perform regulatory functions as well as anchor the dynein to appropriate target sites. Several forms of dynein comprise the outer and inner arms of flagellar and ciliary axonemes, and each is thought to have a distinct role in the beating of flagella and cilia. A cytoplasmic form of dynein is thought to
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(a)
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amino acids 2 000
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move towards the microtubule minusend 13,~4.From recent experiments where the kinesin tail domain was joined to ncd, it was shown that the type of motor domain, and not the tail, determines the direction of movement (R. Stewart and L. Goldstein, pers. commun.). Since the motor domains of kinesin and ncd are very similar to one another (40% amino acid identity), apparently subtle structural changes can have such profound consequences on motor function. It might therefore even be presumptuous to conclude, on sequence information alone, that SMY1 from S. cerevisiae is a microtubulebased and not an actin-based motor. Perhaps in the future, the structural determinants of directionality and other parameters of motor function will be understood, but for now, the functions of motors cannot be inferred simply from their DNA sequences. While sequence information alone does not tell us how motors work, it is valuable for directing new biochemical experiments. As an example, the dynein heavy chain sequence reveals the presence of at least four consensus sites (phosphate binding loops) for nucleotide binding, which was somewhat surprising, since all other known motors (myosin, kinesin, helicases) contain only a single site for ATP binding. One of the dynein sites appears to be where nucleotide hydrolysis takes place, but the functions of the other three sites are unclear. Since these motifs are conserved among several dynein isoforms m sea urchin (I. Gibbons, pers. commun.), it seems likely that the multiple sites serve some important role. Is dynein a four-cylinder motor, or do some
302
of the ATP sites serve an allosteric function? Biochemical investigations will ultimately be required to sort out this problem (perhaps in combination with in vitro mutagenesis), but the sequence information derived from cDNA cloning is responsible for bringing this intriguing question to the fore. Amino acid sequences of the tail domains of kinesin-like proteins have unfortunately been less revealing than those from the 'head' (motor) domain. Although the tail domain of conventional kinesin is highly conserved between human, fly, squid and sea urchin, none of the tail domains of the kinesin-like proteins show any sequence similarity to one another, or to any other known protein sequence. This is somewhat surprising, since some of the kinesinlike proteins seem to be involved in similar functions. For example, CIN8'8 (S. cerevisiae), KIPP 9 (S. cerevisiae), bimC 2° (Aspergillus nidulans), and cut72~ (Schizosaccharomyces pombe) all appear to participate in spindle pole-body separation at the onset of mitosis. An evolutionary relationship between these proteins is suggested by the fact that their motor domains are more highly conserved to one another (~60% amino acid identity) than to other kinesins (30-40% identity). These findings suggest that the tail domains of kinesin motors can diverge considerably during the course of evolution, yet still carry out the same function. It is also possible, however, that the exact homologues of the above mentioned kinesin-like proteins have still not been identified and sequenced from these different species. In addition to providing sequence information, cDNA clones are proving
to be powerful tools for studying the biochemistry of motors. Cytoplasmic motor proteins are present in very low abundance in most cells, which imposes a serious impediment to X-ray crystallography, pre-steady state kinetics determinations and other studies requiring large quantities of pure protein. However, the ability to express active motors in E. coli 12q4 now makes biochemical and functional analysis of any motor a feasible undertaking. Different domains of a motor protein can also be expressed separately for functional or crystallization studies. For example, expressed motor domains can be used to study chemomechanical transduction, and expressed tail domains should be useful reagents for identifying motor receptors. However, since most forcegenerating enzymes contain associated light chains, studying expressed heavy chains alone will not provide a complete understanding of how motors function and are regulated in vivo. Thus, motors also must be biochemically purified in their native state in order to determine their quaternary structure and to study their functional properties.
Motor superfamilies - why so many models to choose from? Deciphering the biological roles of the numerous motors being discovered is an important, although somewhat daunting endeavor. One could imagine that each motor is highly specialized for a unique force-generating task. By such reasoning, the extent of motor superfamilies would reflect the diversity of microtubule force-generating activities that the cell has to perform.
TIBS 1 7 - A U G U S T 1 9 9 2
PylOn Dynamin is a microtubule-activatedGTPaseT M that belongs to a superfamily of proteins sharing homology in the amino-terminalGTP-binding domain3°. Although initially reported to function as a microtul0ule motor, recently it has become uncertain whether this protein possesses force-generating capability. Further work is needed to settle this issue. Dynaminconsists of a single I 0 0 kDa polypeptidethat contains a -300 amino acid amino-terminal globular domain with a consensus GTP-binding site and a carboxy-terminaldo.main that binds to microtubules (see Rg.; J. Herskovitz and R. Vallee, pets. commun.). Dynamin mutations in Drosophila (shibire) produce defects in the endocytosis and recycling of synaptic vesicles at nerve terminals. The rela-
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I~S~ and an E. coil protein i n v o ~ in chromoso~ partitioning (MokB). The common f u s i o n linking these proteins ,et been ascertained, but isan active area of investigation. . . .
Clearly, there is some truth to this idea; however, strictly speaking, a paradigm of 'one task - one motor' is inconsistent with recent data. For example, yeast mutants defective in either of two kinesin-like proteins (KIP1 and CIN8) are viable and have little or no abnormal phenotype; however, the double mutant is not viable due to a defect in the assembly of the mitotic spindle ~8J9. Such findings suggest an overlapping and redundant function of these kinesin-like motors. Individual motors can also perform multiple tasks. For example, mutational analyses show that KAR3, a kinesin-like protein in S. cerevisiae, produces nuclear migration during mating and also performs some as yet undefined function in mitosis. Another example of a multifunctional motor is cytoplasmic dynein, which in mammalian cells has been implicated as a motor for membrane organelles in interphase and chromosome movements during mitosis. The apparent divergence of cytoplasmic motors in various species may also reflect the multitude of ways in which organisms carry out force-generating tasks. For example, the strategies for segregating chromosomes during mitosis and meoisis can vary considerably. In mammalian cells, separation is accomplished primarily by chromosomes moving along kinetochore fibers towards the pole, while in fungi, separation is accomplished primarily by pushing the two spindle poles apart from one another. Some force-generating processes also appear to be rather unique to particular organisms. Examples include microtubule-based delivery of nurse-cell contents to the oocyte during insect embryogenesis or the microtubule-dependent exchange of micronuclei during Tetrahymena conjugation. Thus, although tubulin and
motor domains are highly conserved between species, it seems as though objects can be pushed and pulled in cells in a variety of different ways, which produce equally effective outcomes. Customizing the engine
The force-generating properties of motors also appear to be well adapted to suit the biological context in which they operate. For instance, the transport of membrane vesicles appears to be driven by very few motors, in some instances perhaps by only one. To meet this challenge, kinesin (and probably cytoplasmic dynein) remains bound to microtubules for the vast majority (>99%) of its ATPase cycle22.23;this prolonged attachment time enables single motor molecules to transport organelles for long distances without falling off the microtubule. In contrast, thousands of dynein molecules interact with adjacent outer doublet microtubules in the axoneme, which makes it unnecessary for individual motors to remain persistently and strongly bound to microtubules. Indeed, in vitro motility data (R. Vale, unpublished) seem to confirm that axonemal dynein is strongly microtubule-bound for only a short portion of the hydrolysis cycle. Some motors are also built for speed, while others produce slow and steady movement. The fast beating of cilia and flagella requires that axonemal dyneins produce rapid microtubule sliding (>10 lim sec-~); chromosome motors, on the other hand, operate at 100-1 000-fold slower rates. Some properties of microtubulebased motors are also so remarkable that they seem to challenge conventional ideas of how chemomechanicai enzymes work. Certain inner arm axonemal dyneins, for example, rotate microtubules about their longitudinal
axis as they move them forward across a glass surface, indicating that they produce torque as well as a forward propulsion 24. Such movement is difficult to explain without evoking some modification of the traditional swinging crossbridge model, which envisages that the motor changes its angle relative to the filament and produces movement by an oar-like mechanism. Even more surprising and difficult to explain mechanistically is the finding that kinesin and the kinesin-like motor ncd move in opposite directions along the microtubule ~3,14. Perhaps, by understanding what specifies the directon of movement, one may also get at the heart of the chemomechanical transduction mechanism itself.
Outlook Eight years ago, progress in cytoplasmic motility was thwarted by an almost complete lack of information on intracellular protein motors. Today, with a number of motors known and reagents available (e.g. cDNA clones, antibodies), it is relatively straightforward to identify a variety of kinesins (and in the near future dyneins) from almost any eukaryotic organism. The greater challenge now comes in deciphering the logic of how organisms use their multitude of force-generating machines and in understanding how motors produce movement from chemical energy. Although these problems are difficult, the problem of microtubule motors has brought together an excellent group of scientists with diverse backgrounds and expertise. Individuals, who eight years ago were studying worm development, axonal transport or the kinetics of the myosin ATPase, now find themselves studying the same collection of microtubule motors. If information gathering
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continues at the present rate, the world of cytoplasmic motility will be very different again by th$ time the 300th issue of TIBS is published.
References 1 Endow, S. A. ($991) Trends Biochem. Sci. 16, 221-225 2 Goldstein, L. S. B. (1991) Trends Cell Biol. 1, 93-98 3 Vale, R. D. and Goldstein, L. S. B. ($990) Cell 60, 883-885 4 Bloom, G. S. (1992) Curr. Op. Cell Biol. 4, 66-73 5 Porter, M E. and Johnson, K. A. (1989) Annu. Rev. Cell Biol. 5, 119-151 6 Schroer, T. A. and Sheetz, M. P. (1991) Annu. Rev. Physiol. 53, 629-652 7 Vallee, R. B. and Shpetner, H. S. (1990) Annu. Rev. Biochem. 59, 909-932 8 Allen, R. D. (1985) Annu. Rev. Biophys. Biophys. Chem. 14, 265-284 9 Inoue, S. (1986) Video Microscopy, Plenum 10 Allen, R. D. et al. (1985) J. Cell Biol. 100, 1736-1752
11 Vale, R. D., Schnapp, B. J., Reese, T. S. and Sheetz, M. P. (1985) Cell 40, 559-569 12 Yang, J. T. et al. (1990) Science 249, 42-47 13 McDonald, H. B., Stewart, R. J. and Goldstein, L. S. (1990) Cell 63, 1159-1165 14 Walker, R. A., Salmon, E. D. and Endow, S. A. (1990) Nature 347, 780-782 15 Lillie, S. H. and Brown, S. S. (1992) Nature 356, 358-361 16 Gibbons, I. R., Gibbons, B. H., Mocz, G. and Asai, D. J. (1991) Nature 352, 640-643 17 Ogawa, K. (1991) Nature 352, 6 4 3 4 4 5 18 Hoyt, M. A., He, L., Loo, K. K. and Saunders, W. S. J. Cell Biol. (in press) 19 Roof, D. M., Meluh, P. B. and Rose, M. D. J. Cell Biol. (in press) 20 Enos, A. P. and Morris, N. R. (1990) Cell 60, 1019-1027 21 Hagan, I. and Yanagida, M. (1990) Nature 347, 563-566 22 Block, S. M., Goldstein, L. S. and Schnapp, B. J. (1990) Nature 348, 348-352 23 Howard, J., Hudspeth, A. J. and Vale, R. D. (1989) Nature 342, 154-158 24 Vale, R. D. and Toyoshima, Y. Y. ($988) Cell 52,
459-469 25 Ferreira, A. et al. (1992) J. Cell Biol. 117, 595-606 26 Hollenbeck, P. J. and Swanson, J. A. (1990) Nature 346, 864-866 27 Pfister, K. K. et aL ($989) J. Cell Biol. 108, 1453-1464 28 Saxton, W. M., Hicks, J., Goldstein, L. S. B. and Raft, E. C. (1991) Cell 64, 1093-1102 29 Hackney, D. D., Levitt, J. D. and Suhan, J. (1992) J. Biol. Chem. 267, 8696-8701 30 Obar, R. A., Collins, C. A., Hammarback, J. A., Shpetner, H. S. and Vallee, R. B. (1990) Nature 347,256-261 31 Shpetner, H. S. and Vallee, R. B. (1992) Nature 355, 733-735 32 Stewart, R. J., Pesavento, P. A., Woerpel, D. W. and Goldstein, L. S. B. (1991) Pro& Natl Acad. Sci. USA 88, 8470-8474 33 Endow, S. A. and Hatsumi, M. (1991) Proc. Natl Acad. Sci. USA 88, 4424-4427 34 Cole, D. G. et al. (1992) J. Cell Sci. 101, 291-301 35 Sawin, K. E., Mitchison, T. J. and Wordeman, L. G. ($992) J. Cell Sci. 101, 303-313
OBITUARY Peter Mitchell 1920-1992
the first clear statement of the electroOnly rarely do individual scientists leave chemical proton gradient as the an indelible mark on a field. Peter coupling intermediate, the need for a Mitchell made such a mark when he membrane with low proton permeability, proposed and then developed and and the mechanism by which dinitrodefended the chemiosmotic theory of phenol uncouples oxidative phosphorylenergy coupling in oxidative phosation. None of these proposals was supphorylation, photophosphorylation and ported by experimental evidence at the active transport systems. time, as illustrated by the fact that the Mitchell obtained his PhD in biooriginal hypothesis had the direction of chemistry from Cambridge, with J. F. Danielli, and particularly appreciated proton movements backwards. his contact there with David Keilin. He Experimental work by Mitchell and Jennifer Moyle soon provided the evilater moved to the University of Edindence that mitochondria do transport burgh, and, in 1963, moved to a ruined protons in the manner proposed, but it great house near Bodmin, Cornwall. took about ten years to convince the Mitchell designed the restoration of majority of biochemists that the theory Glynn House to contain his family apartments and research laboratories, and was fundamentally correct. Mitchell's worked on the project for two years characterization of this evolution in scientific thinking was typically gracious: alongside the craftsmen. Mitchell and 'most remarkable and admirable is the his brother then endowed the Glynn altruism and generosity with which the Research Foundation with £240000 of their inheritance, creating an institution former opponents of the "chemiosmotic which is still very active. hypothesis" not only came to accept it, In part, his work reflected a conbut actively promoted it to the status of scious effort from his student days to membranes by electron transfer as an a theory'2. bridge a gap between scientific fields. integral part of respiration. Mitchell We can safely say that Mitchell's Chemical reactions belonged to chem- extended this idea to include a proton work revolutionized biochemistry and istry, electric fields belonged to phys- transporting ATPase, thus explaining transport physiology, but Mitchell did ics, and processes containing elements ATP synthesis in mitochondria and not believe that science was advanced of both were likely to be ignored. The chloroplasts. But the brilliance of his by revolution: 'let me hasten to remark plant physiologists E. J. Lund and H. proposal came from the completeness ... that I make no claim to biochemical Lundegardh had proposed that electric of its construction. His paper in Nature ~ originality, except, perhaps, inasmuch fields are generated across biological not only proposed the ATPase, but was as I have consciously endeavored to © 1992,ElsevierSciencePublishers,(UK) 0376-5067/92/$05.00 304
