Functions of microtubule-based motors.

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FUNCTIONS OF MICROTUBULE-BASED MOTORS Trina A. Schroer] Michael P. Sheetz2 Department of Cell Biology and Physiology, Washington University Medical School, St. Louis, Missouri KEY WORDS:

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kinesin, cytoplasmic dynein, vesicle transport, motility

INTRODUCTION Energy-dependent transport of macromolecules along microtubules is the basis for a variety of intracellular processes including vesicle transport, mitosis , and the motility of cilia and flagella. This review focuses primarily

on microtubule-based transport of membrane vesicles. It is becoming appar ent that microtubule-based transport is important in the pathways of mem brane biogenesis and recycling, and in some cases specific roles for the microtubule-dependent mechano-enzymes, kinesin and cytoplasmic dynein, have been proposed. In this article we review recent work in the field and

summarize what is known about the role of microtubule-based motility in vesicle transport.

MICROTUBULE-BASED TRANSPORT Functions A fundamental teleological question is why a cell would utilize energy (ATP) to deliver materials to their intracellular destinations rather than relying on

IDepartsnent of Biology, The Johns Hopkins University, Baltimore, Maryland

21218 2Department of Cell Biology, Duke University Medical Center, Durham, North Carolina

.

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diffusion. When we consider the movement of chromosomes that occurs in mitosis, the answer seems clear. Chromosomes are large objects (1-20 JLm long) that would be expected to have a low diffusion coefficient in cytoplasm (D 10-10_10-12 cm2/sec). During cell division, daughter chromosomes are separated from one another and a complete set of chromosomes is delivered to each daughter cell. The microtubules of the mitotic spindle provide the structural support for this process and are absolutely required for chromosome alignment, segregation, aJ;ld subsequent delivery to the two daughter cells, all steps that require ATP hydrolysis. Thus mitosis requires that chromosomes be moved in an energy-dependent, directed process. This is the essential role played by all varieties of microtubule-based intracellular transport. The transport of membrane vesicles within neuronal axons and dendrites requires energy for the simple reason that diffusion does not allow rapid transport over long distances (i.e. hundreds of mm). Moreover, neuronal viability requires the highly directional anterograde transport of material to the synapse, as well as the retrograde transport of material to the cell body. In smaller, less highly elongated cells, the functions served by microtubule based vesicle transport are less well understood. Most intracellular vesicles need only travel short distances « 20 /Lm) at rates (10 /Lm in five min) that could result from diffusion through an aqueous medium. Cytoplasm is a viscoelastic liquid, however, that does not allow free diffusion of large molecules (> 20-40 nm in diameter). The intracellular cytoskeleton allows diffusion at rates inversely proportional to particle size (71). These studies predict that a vesicle larger than 50 nm in diameter (Mr 1 x 108) would diffuse at an extremely slow rate and might not even have access to certain regions of the cytoplasm. An active transport mechanism would greatly facilitate the movement of these vesicles through the cytoplasm. We will discuss the possible functions for microtubule-based motility in intracellular mem brane traffic. For example, the behavior of early endocytic and late exocytic vesicles is not affected if cells are treated with microtubule inhibitors (see below). In contrast, microtubules seem to facilitate membrane export (1,29; G. van Meer, K. Simons, personal communication), perhaps by directing membrane vesicles to their destinations (95). Likewise, microtubules may facilitate membrane traffic through the endocytic pathway. The positioning of the Golgi apparatus and lysosomes within cells also depends on an intact microtubule cytoskeleton (19, 45, 75). What is less clear is the extent to which these processes continue in the absence of microtubules and whether there are membrane transport processes that absolutely depend on microtubules . In summary, the functions of energy-dependent transport are (a) to provide the force to move large intracellular components, (b) to deliver and con centrate these components at specific locations, and (c) to transport these =


SCHROER & SHEETZ

MICROTUBULE-BASED MOTILITY

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Functions of Microtubule Based Motors �


•

Force Generation

Figure 1

MT

:1=-8

Concentration

This diagram illustrates the three major functions of the microtubule-based motility in

cells: force genenltion, concentration, and transport. Examples of force generation are the separation of chromosomes during mitosis and the extension of the endoplasmic reticulum (ER) network. Membranes become concentrated in the synapse by microtubule-based transport from the cell body through the axon over long distances. Similarly, the Golgi apparatus remains concentrated in the centrosomal region by transport along microtubules that have their minus ends anchored at the MTOC

(45). In the insect ovariole, yolk granules are transported along microtu

buies to the egg (l lO).

components faster and over longer distances than allowed by diffusion (Figure I), Microtubule Organization A microtubule is a head-to-tail polymer of asymmetric subunits, hence each microtubule filament has an intrinsic polarity. The two ends of the filament are termed the plus and minus ends based on relative rates of tubulin polymerization (the plus end supports rapid polymerization, whereas the minus end polymerizes at a slower rate). This polarity also directs the mechanochemical motors that have specific polarity preferences (either plus end or minus end directed; see below). The arrangement of microtubules within a cell will therefore determine the direction of motor-based transport. Microtubule polymerization is nucleated at intracellular sites aptly named microtubule organizing centers (MTOCs); most cells contain a single MTOC, which provides a unique site for polymerization. Microtubules polymerize from the MTOC with their plus ends distal; in fibroblasts, for example,


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SCHROER & SHEETZ

microtubules radiate from a site near the nucleus toward the periphery, The MTOC is usually coincident with the centrosome; however, some interesting exceptions to this rule should be noted (Figure 2), In Madin-Darby canine kidney (MDCK) cells, a polarized epithelial cell line, most of the microtu buIes are found in bundles with the minus ends nearest the apical surface and plus ends near the basal surface; therefore the MTOCs would be expected to be diffuse and positioned in the apical domain (9), It will be of great interest to determine whether a similar arrangement occurs in other epithelial cells. In cardiac myocytes (in culture) microtubules are arranged in the typical con figuration with plus ends radiating toward the periphery; however, polymerization appears to be initiated at multiple sites around the cell nucleus (65). Microtubules in nerve cell axons are oriented with plus ends toward the periphery. In contrast, the microtubules in dendrites can be arranged with either minus ends or plus ends outward (8, 20). Dendrites support a form of vesicle transport that is analogous to fast axonal transport (34), but it is difficult to imagine how directional transport can occur on an antiparallel mixture of microtubules. It is possible that microtubules of one polarity are somehow modified so that they are not recognized by the microtubule motors (108). This modification might be mediated by a microtubule-binding protein that inhibits motility, for example, by microtubule-associated protein 2 (MAP 2) (89).

Microtubule Organization

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Figure 2

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Three basic types of microtubule organization

Linear are

illustrated in this diagram. In

fibroblasts, the microtubules are typically arrayed radially from a single MTOC, which co localizes with the centrosoipe. In epithelial cells (speCifically in MDCK) there are multiple MTOCs in the apical region of the cell that produce a parallel arra y of microtubules stretching from the apical to the basolateral surface (9). A third pattern is found in the axon where microtubules

are

aligned in an overlapping linear array with a single polarity

(21).

MICR01UBULE-BASED MOTILITY

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KD

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}-

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Figure 3 This polyacrylamide gel shows the polypeptide compositions of the two purified motors, kinesin (K) and cytoplasmic dynein (D). The kinesin alpha subunit is 116 kd and the beta subunit is about 60 kd. For cytoplasmic dynein the subunit molecular weights are 440 (HC), 70 (IC), and SO (LC) kd.

Microtubule-Based Motors

The microtubule-based mechano-enzymes (motors), kinesin and cytoplasmic dynein, are discussed at length elsewhere (80,120,121). Both are ubiquitous and are present in high concentrations intracellularly. The motors are microtu bule-activated ATPases that can drive the gliding movements of microtubules on glass coverslips; kinesin causing movement toward the plus ends of microtubules, and cytoplasmic dynein causing movement toward the minus ends of microtubules. Kinesin is a heterotetramer, Me = 350,000, composed of two a chains (Me = 116,000) and two f3 chains (Me = 62,000; Figure 3). The amino acid sequence of the a chain has been determined (63, 133). This polypeptide appears to contain three structural domains; an N-terminal head, which contains the microtubule- and ATP-binding sites (this has been termed the motor domain; 121); a filamentous rod domain; and a tail domain that is believed to be the site for the binding to membrane vesicles or other struc-


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SCHROER & SHEETZ

tures. The predicted structure corresponds well to the kinesin ultrastructure defined by rotary shadow electron microscopy and epitope mapping studies (6, 43, 44, 102). Cytoplasmic dynein (Mf = 1,200,000) is composed of two heavy chains (Mr = 440,000) and multiple intermediate (Mr = 68,000-72,000) and light chains (Mr = 50,000-55,000; Figure 3). The ultrastructure of cytoplasmic dynein is well characterized; the molecule resembles a flower pot with two blooms (111, 124). Cytoplasmic dynein has two large, spherical heads con nected to a compact base; the base is composed of at least four globular subdomains. Filaments are occasionally observed projecting from the spheri cal heads, but they are not a consistent structural feature of the molecule. Studies correlating the heavy, intermediate, and light polypeptide chains with specific structural domains should soon be possible because of the recent development of monoclonal antibodies against the cytoplasmic dynein sub units (112). Determination of the primary sequences of the different dynein subunits is no doubt forthcoming. A family of proteins that share primary sequence homology with the kinesin a chain has recently been discovered [yeast KAR3 (81); Drosophila ned (30, 79)]. These kinesin-like proteins have significant homology with the kinesin motor domain, but in contrast to authentic kinesin, the motor domain is found at the C-terminus. The N-terrnini bear no apparent homology to kinesin and are presumed to be involved in attaching the protein to the object it moves (vesicle, chromosome, microtubule, or other). Aspergillus also contains a kinesin-like protein, but in this case the motor domain is at the N-terrninus and the protein diverges significantly at the C-terrninus. The kinesin-like proteins are reminiscent of the myosin Is, a family of actin binding motors that are encoded by at least five distinct genes in Dietyo stelium (117). The actin- and microtubule-based motors share several features; the direction of movement is determined by the filament substrate, and both classes of motors have similar ATPase cycles ,39, 54). What distinguishes the motors from one another are the filament substrates, the direction of movement, the objects moved by each, and the mechanisms for regulation of movement. The Organelle Motor Complex

Kinesin and cytoplasmic dynein by themselves can convert the energy derived from ATP hydrolysis into movement, as evidenced by the fact that they cause microtubule gliding. One might expect that the enzymes would be competent to drive organelle motility as well. It is now apparent that kinesin and dynein alone cannot drive the movement of membrane vesicles and that other soluble proteins play a key role (104, 105). It is also clear that membrane proteins are required (103, 123). It has been proposed that the motors interact with the



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organelle surface as part of a larger protein complex that has been called the organelle motor complex. This includes the motor, a membrane-associated motor receptor, and the other soluble components required for motility (108). We have identified two distinct soluble accessory factors that stimulate vesicle movement (T. Schroer, M. Sheetz, manuscript in preparation), and a potential candidate for the membrane protein that binds kinesin (the kinesin receptor) has been found in brain microsomes (I. Toyoshima, M. Sheetz, manuscript in preparation). Further work is required to define how the components of the organelle motor complex interact to regulate vesicle transport. EXPERIMENTAL APPROACHES Pharmacological Studies

Pharmacological agents that affect microtubule polymerization are commonly used to determine the role(s) played by microtubules in different intracellular processes. These studies utilize the microtubule-depolymerizing drugs, col chicine, co1cemide, and nocodazole, or the microtubule-polymerizing drug, taxol. As is often the case with pharmacological studies, the results of these experiments must be interpreted with caution. Non-specific effects of the drugs on processes that do not involve microtubules are common and must be determined before strong conclusions can be drawn. For example, a microtu bule poison may bind to and inhibit a non-microtubule protein; this has occurred in the case of colchicine (60). Fortunately, lumicolchicine (a photo inactivated form of colchicine) can be used to determine colchicine's non specific effects. It is useful to determine whether a process is inhibited by more than one anti-microtubule drug. Recent studies have found inhibitory effects whose magnitudes correlate well with the ability of each drug to induce microtubule depolymerization (87). Microtubule-depolymerizing agents act by binding to depolymerized tubulin and blocking its assembly into tubulin polymer, consequently, only microtubules that are dynamic (i.e. those undergoing continual polymerization and depolymerization) are affected; sta bilized microtubules are not induced to depolymerize. Microtubule poisons commonly leave intact a subset of cytoplasmic microtubules that may be competent to support vesicle transport. A final complication to the pharmaco logical approach arises because microtubules contribute significantly to cytoplasmic viscosity which, as described above, profoundly affects the rate of intracellular diffusion. Despite these caveats, pharmacological studies have contributed significantly to our understanding of the role played by microtu buIes in intracellular motility. In general, most agents that inhibit kinesin (i.e. NEM, AMP-PNP, and vanadate) inhibit cytoplasmic dynein and vice versa; moreover these agents

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SCHROER & SHEETZ


affect a myriad of other cellular proteins and functions. One pharmacological treatment exists that is selective for dyneins, vanadate-mediated ultraviolet cleavage (32, 72, 90, 92). UV-photocleavage of dynein selectively inhibits minus-end directed vesicle movement in vitro (100, 105). This treatment has not been used in studies of motility in vivo because vanadate does not readily permeate intact cell membranes. Genetics

The combined use of classical and modem molecular genetic techniques makes the genetic approach a particularly powerful means for studying pro tein function in vivo. Genetic analyses of kinesin and cytoplasmic dynein function are currently being performed in several organisms including the budding yeast (Saccharomyces cerevesiae), Aspergillus, Dictyostelium, Dro sophila, and C. elegans. A common approach is to clone and sequence a motor gene, then use the deduced protein sequence to design and create mutations in the gene of interest. The mutant gene is then reintroduced into the organism, and the phenotype is determined. For example, if the gene encoding the heavy chain of kinesin is deleted from Drosophila, the resulting flies do not survive to adulthood; the phenotype of early embryos and larvae suggests defects in nerve cell function (98). This finding is consistent with the hypothesis that kinesin plays an essential role in fast axonal transport. Kinesin and cytoplasmic dynein were recently isolated from Dictyostelium (61, 78), an organism that has been used extensively in analogous genetic studies of myosin function (116). Once the genes for the Dietyostelium microtubule motors are cloned, it will be possible to perform gene disruption experiments to determine the roles they play in vesicle motility, mitosis, and other microtubule-based motile behaviors. This line of investigation is likely to yield a wealth of information in the near future. Another type of genetic analysis of motor function relies heavily on serendipity. The starting point is a previously described mutation. When the mutant gene is cloned and sequenced, it is discovered to have primary sequence homology with a motor. This approach was the basis for the discovery of the aforementioned kinesin-like proteins ned in Drosophila, KAR3 in yeast, and bimC in Aspergillus. Mutations in the Drosophila ned gene result in defective meioses and early embryonic mitoses (30, 79). KAR3 mutants (81) demonstrate incomplete karyogamy, the process by which parent haploid nuclei migrate toward each other and fuse to form a diploid nucleus. Aspergillus bimC mutants cannot complete mitosis (31). In summary, genetic studies suggest that kinesin and its homologues drive a variety of intracellular transport events. It is highly likely that cytoplasmic dynein-like proteins exist to fulfill a complementary set of transport functions. Discovery of dynein homologues will have to wait until the primary sequence of the motor domain of cytoplasmic dynein is known.


637

In Vitro Assays The development of an in vitro assay for microtu bule-based transport was a major breakthrough that led to the discovery and characterization of the motor activities of kinesin and cytoplasmic dynein. The movement of vesicles on microtubules has also been reconstituted from isolated components, thus making it possible to define the soluble factors required for transport. As described above, kinesin and cytoplasmic dynein provide the force for plus-end and minus-end vesicle transport, but both require additional accessory factors to move vesicles (104, 105). Multiple soluble factors that activate motor-driven vesicle transport have been identi fied (T. Schroer, M. Sheetz, manuscript in preparation). The reconstituted assay for vesicle transport should facilitate the identification and purification of membrane receptors for kinesin and cytoplasmic dynein and will be useful in future studies of the regulation of vesicle motility.


VESICLE TRANSPORT

It has not been possible to reconstitute the entire process of mitosis in vitro because of the complexity of the mitotic apparatus. The presently available in vitro systems mimic individual mitotic phases. Mitchison & Kirschner (82) bound isolated chromosomes to centrosome microtubules; under certain experimental conditions the chromosomes moved toward plus and minus ends of microtubules in a manner reminiscent of prometaphase (52). A similar assay reconstituted the movement of chromosomes toward the centrosome (analogous to poleward movement in anaphase A; 62). Others have isolated metaphase-arrested mitotic spindles from diatoms and fission yeast (Schizosaccharomyces pombe); the spindles can be reactivated in vitro to enter into and complete anaphase (74). Further effort should lead to the identification of components of the chromosome and the spindle microtubules that contribute to mitotic motility.

MITOSIS

Antibody Inhibition Studies Antibody probes are commonly used as a means for determining the function of a protein. The antibody is introduced into the experimental system, then various experimental parameters are measured to determine if the antibody has an effect on the process of interest. This approach was used to determine the effects of anti-motor antibodies on vesicle transport in squid axoplasm. Antibodies against either dynein or kinesin inhibited motility in both the anterograde and retrograde directions [anti-dynein antibody (33); anti-kinesin antibody (17)]. These studies must be interpreted with caution because intact antibody molecules (not Fab fragments) were used at high concentrations. The antibodies may have cross-linked vesicles and microtubules to produce a physical block to movement.

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SCHROER & SHEETZ

MICROTUBULE-BASED VESICLE TRANSPORT


Gen,eral Features Early light microscopic observations of living cells revealed that cytoplasm contains a significant number of moving particles; however, these studies were limited by the fact that only particles larger than 200 nm in diameter were visible (reviewed in 93). This population of large membrane-bound or�anelles includes mitochondria, lysosomes, and secretory granules, but not the smaller vesicles that mediate anterograde axonal transport and many other membrane traffic events. The technique of video-enhanced light microscopy (3, 53, 99) made it possible to study the movements of extremely small vesicles (50-80 nm) and allowed fast axonal transport in the anterograde direction to be observed for the first time (4, 16). In recent years the video microscope has been turned on a wide variety of cells to demonstrate that, in general, cytoplasm is teeming with moving particles (vesicles). In the major ity of cells vesicle motility is microtubule-based; vesicles move along linear paths that suggest microtubules and motility can be blocked by microtubule depolymerization. Moving vesicles display a spectrum of behaviors. Some undergo bidirectional saltatory movements, while others move unidirectional ly over great distances (> 10 J,tm). In extremely flat regions of cells it is possible to observe vesicle movements on single microtubules (119) and the motility of a tubulovesicular membrane system (S. Dabora, M. Sheetz, unpUblished) that behaves like the endoplasmic reticulum (ER) (67). Differ ential interference contrast microscopy does not indicate the identities of membranes, but certain intracellular membranes can be specifically labeled with fluorescent probes. The ER can be stained with DiOC6 (3) (115), Golgi membranes become labeled with NBD-ceramide (70), mitochondria stain with rhodamine 123 (55), and lysosomes and other low pH compartments accumulate acridine orange (10). It is also possible to tag intracellular mem branes with fluorescently labeled antibodies introduced by microinjection (7). These studies have revealed that the ER, Golgi membranes, secretory gran ules, and endocytic vesicles undergo extensive transport (23, 28, 45, 64). Additional probes that are specific for different intracellular compartments will be useful in future studies of vesicle motility.

Fast Axonal Transport Fast axonal transport is the archetype of microtubule-based vesicle transport (5, 101), and much of our understanding of its molecular basis has come from axonal systems (57, 108). Nerve cell axons are highly elongated processes that must support the transport of membranous organelles at rates and over distances far greater than can occur by diffusion. Fast transport is un equivocally an energy-dependent (2), microtubule-based process (5, 101).



639

Since axonal microtubules are uniformly aligned with their plus ends distal to the cell body, anterograde fast transport would be expected to be driven by kinesin and retrograde fast transport by cytoplasmic dynein. This has been demonstrated by reconstituted studies of axonal transport in squid axoplasm (100, 104). Although the motors for fast transport are now identified, a number of questions regarding the mechanism of fast transport remain unanswered. Some progress has been made toward identifying the accessory factors re quired by kinesin and dynein (T. Schroer, M. Sheetz, manuscript in prepara tion), but it is not known how the factors regulate motor activity. A significant problem arises from the fact that protein synthesis occurs only in the nerve cell body (soma) so all proteins required at the synapse must be delivered there by anterograde transport. Specifically, how does cytoplasmic dynein reach the synapse? It has been proposed that dynein is transported on vesicles, presumably in association with its normal receptor (l08). After arrival at the synapse, the anterogradely transported membrane returns to the cell body in the form of a retrograde transport vesicle. The switch from anterograde to retrograde must occur at the synapse. The problem of directionality switching is not limited to neurons. As a bit of membrane moves through the biosynthetic and recycling compartments, it is subject to both plus-end and minus-end directed motility. An example of this is the behavior of the poly-Ig receptor (pIgR) in polarized epithelia. The pIgR is synthesized and processed in the ER and Golgi apparatus and is then delivered to the basolateral plasma membrane. After binding ligand (poly-Ig) at the cell surface, the receptor is endocytosed and transported across the cell to the apical surface. This process is known as transcytosis (66, 84), and it has been demonstrated to rely on intact microtubules (see below). During the course of its life cycle, the pIgR first appears in vesicles that move by diffusion and later in vesicles that are competent to be transported on microtu buIes. Clearly the cell must be able to distinguish naked pIgR from ligand bound pIgR to transport the receptor appropriately. A completely novel mechanism for directionality control was recently postulated by Heuser (42), who observed that the direction of movement of endosomes would change in response to changes in cytoplasmic pH. A more complete understanding of the organelle motor complex is needed before the molecular events that control vesicle directionality can be defined. It is also unclear why the microtubule-based motors should be present in such high concentration in axons. Both kinesin and cytoplasmic dynein are abundant soluble proteins (46, 112), yet only one or a few motor molecules per vesicle are required to drive transport (48). This translates into a 1,0005,OOO-fold excess of motors over vesicles in the axoplasm (104). Motor activity must be tightly regulated, most likely by the components of the


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SCHROER & SHEETZ

organelle motor complex. It is possible that the interaction of motors with their membrane receptors is highly dynamic (lOS). This might explain the variable amounts of vesicle staining observed in kinesin immunolocalization studies (46, 85, 91). No matter how dynamic the kinesin-vesicle interaction, kiriesin is transported rapidly in axons, which suggests that it is associated with membranes (83). A newly synthesized vesicle in a neuron can be transported through the axon or the dendrites. Since different proteins are targeted in the two direc tions, a sorting mechanism must exist to ensure that materials reach the proper destination. The microtubules within dendrites are arranged with = 50% plus end distal and = 50% minus end distal, which may influence motility (8,20). Vesicle transport may also be affected by MAPs that have different distribu tions in axons and dendrites (reviewed in 77). Because the direction of transport is determined by the polarity of microtubules, and because moving vesicles can switch between adjacent microtubules, it is difficult to imagine a mechanism for unidirectional vesicle transport on a mixed microtubule sub strate. One subset of microtubules might be coated with a MAP to activate or inhibit vesicle motility; this would create a microtubule array that was effec tively unidirectional. Vesicle transport in dendrites raises several important questions that can only be answered by further study.

Tubular Membrane Systems Certain intracellular membranes adopt an extended tubular conformation. These include the ER (67, 68, 114), the ER-Golgi intermediate compartment (69), the trans-Golgi network (TGN) (35), endosomes (42, 73) and, in some cases, lysosomes (113). In some cases, the distribution of membrane corre lates with the distribution of microtubules. Our understanding of the mech anisms that drive tubule extension, motility, and fusion is based largely on in vitro studies. Large membrane aggregates were observed to spread out into interconnected networks in an ATP-, motor- and microtubule-dependent man ner (24; V. Mermall et aI, manuscript in preparation). Initially, small mem brane buds emerged from the aggregates and became elongated as they were pulled along microtubules. Intersecting tubules fused and branched into poly gonal shapes. The motility process has been called microtubule-dependent tethering to emphasize its similarity to the biophysically characterized process of tether formation (129). Similar membrane tubules are also formed as a result of the movement of microtubules along the substratum (122), by the movement of a myosin-like motor on actin filaments (56), or simply by focal application of force to a membrane bilayer (128). The behavior of membrane networks in vitro bears a striking resemblance to behavior of the ER in live cells where motility occurs on stationary microtubules (67, 68). The dynamic morphology of the ER is dependent on the microtubule-based


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motors (V. Mermall et aI, manuscript in preparation). In contrast, other tubular membranes (such as lysosomes) may be formed from the fusion of smali vesicles. Microinjection of inhibitory anti-kinesin antibodies causes tubular lysosomes to collapse into the cell center, which suggests that kinesin is involved in tubule extension

(47). Similar studies should elucidate the role


of microtubule-based motility in the function and motility of other membrane tubule systems.

MEMBRANE TRAFFIC AND MICROTUBULES Pathways of Membrane Traffic Membrane vesicles mediate the transport of proteins and lipids between the biochemically distinct compartments that comprise the pathways of mem brane biogenesis and recycling. Several steps have been proposed to involve microtubule-based motility. Although microtubules are clearly required for vesicle transport over long distances (i.e. in neurons) the small size of most cells

(10--50 JLm diameter) makes the need for a microtubule-dependent

transport mechanism less obvious. Still, cytoplasmic vesicles may be im mobilized by their interactions with the cytoskeleton or other membranes so that microtubule-based transport is required for movement. Alternatively, kinesin and cytoplasmic dynein may facilitate vesicle movements that would otherwise occur by diffusion, albeit at a reduced rate. In the first case, inhibition of microtubule-based transport should block the process altogether. In the second case, inhibition of transport would be expected to reduce the rate of membrane traffic, but not its overall extent. In order to distinguish between these possibilities, it is necessary to assay the process both im mediately after inhibition and at much later times. Most studies evaluating the role of microtubules in membrane traffic utilize microtubule inhibitors; not surprisingly the findings are controversial, and conflicting results are often obtained. Better, more specific, inhibitors are needed, but despite the prob lems associated with working with microtubule poisons, a consensus picture is slowly emerging. It is generally agreed that endocytosis (membrane invagination and vesicle budding) and exocytosis (the fusion of vesicles with the plasma membrane), the first and last events in membrane recycling and biogenesis, continue normally in the presence of microtubule poisons. Transport of materials between the cis, medial, and trans compartments of the Golgi apparatus is also microtubule-independent

(95). Similarly, transfer of material between

early endosomal vesicles (before reaching the common sorting compartment where these materials diverge) does not require microtubules

(26, 36). None

of these events involves vesicle movements farther than a few microns. Microtubule-based transport seems to play at least one major role in the


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SCHROER & SHEETZ

secretory and endocytic pathways common to all cells (reviewed in 58). The events that are consistently inhibited by microtubule depolymerization are (a) the recycling of membrane from the cis-Golgi region to the ER, and (b) the movement of vesicles between early and late endosomes (see below). Translocation of material from the basolateral to the apical surface of polar ized epithelia (transcytosis) also appears to depend upon microtubule-based transport. As we will discuss below, treatment of most cells with anti microtubule agents does not significantly inhibit movement of membrane and secretory proteins from the site of synthesis (the ER) to the plasma membrane. Early Events in Membrane Biogenesis: ER Through Golgi

Our understanding of the mechanism of synthesis and export of membrane and secreted proteins ever increases, yet it remains a mystery how the process occurs efficiently, considering the complex three-dimensional structures of the organelles involved. The ER and Golgi apparatus are highly structured organelles whose unique morphologies depend on intact cytoplasmic microtu buIes (19, 45, 114) (see Figure 4). In cells in culture, the ER is a highly extended network of membrane tubules that undergoes continuous microtu bule-based rearrangements (67, 68). The amount of membrane traffic from the ER to the Golgi apparatus is so great that the entire fluid volume of the ER can be transferred to the Golgi in less than 15 min (130). Obviously the ER would become rapidly depleted of membrane without a highly efficient recycling system. A recycling mechanism has been proposed to mediate the retention of proteins that are normally resident in the ER (127; reviewed in 59). Recycled membrane passes through a compartment lying between the ER and cis-Golgi (the "salvage" compartment) that is enriched in two markers, a 53-kd protein (106) and the receptor for the peptide signal (KDEL) that governs protein retention in the ER (125). Under certain conditions the salvage compartment accumulates and can be observed to extend tubular processes (125) in a microtubule-dependent manner (69) (Figure 4). This suggests that membrane recycling from the transition compartment to the ER is mediated by microtubule-based motility. Since the Golgi apparatus and transition compartment lie in the center of the cell and the ER is highly spread, movement toward the ER is expected to be driven by kinesin, the plus-end motor. Membranes of the trans-Golgi network have also been observed to extend tubular processes that undergo microtubule-based movements (23), thus indicating that these membranes are also competent to move on microtubules. Depolymerization of microtubules, either pharmacologically or during mitosis, causes the Golgi apparatus to vesiculate and disperse (19). When the microtubules are allowed to repolymerize, Golgi vesicles are observed to move in a saltatory fashion toward the MTOC, i.e. toward the minus ends of microtubules. The motility


643

Membrane Traffic


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This diagram illustrates the basic roles for microtubule-based motility in the in

tracellular traffic of membranes. The ER network is formed by the movement of membrane strands on microtubules (68), and recycling of membranes from the transition compartment back to the ER is also microtubule-dependent (69). Golgi assembly at the microtubule organizing center (MTOC) appears to depend on cytoplasmic dynein (45), and elements of the trans-Golgi network move along microtubules (23). In some cases, secretory vesicle movements may depend upon microtubules (118). In the endocytic pathway the transition from the early to late endosome is microtubule-dependent (36) and, in some cases, recycling vesicles move toward the MTOC before returning to the plasma membrane. Finally, Iysosomes can align along microtubules (113).

of Golgi-derived membranes is obviously complicated and mechanisms for its control are not yet understood.

Late Events in Membrane Biogenesis: TGN to the Plasma Membrane As described above, the process of fast axonal transport allows the export of newly synthesized membranes and their contents from the cell body toward the periphery. By analogy, membrane secretion in all cells probably relies on microtubule-based motility. In the majority of cells, treatment with microtu-


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bule depolymerizing agents does not inhibit the delivery of material to the plasma membrane. For example,the amount of a viral glycoprotein (VSV G) arriving at the surface of fibroblasts is not reduced after treatment with colchicine (95). Disruption of microtubules seems to alter the distribution of VSV G on the cell surface, which suggests that microtubules normally target secretory vesicles to particular regions of the cell. In cells specialized for secretion, the effect of microtubule depolymeriza ' tion is more profound. The general consensus is that secretory materials synthesized and packaged in the presence of microtubule inhibitors' are not secreted efficiently, whereas secretory granules formed before drug treatment release their contents normally (15, 22, 1 31). A simplistic explanation is that microtubules mediate transport of newly synthesized secretory vesicles to the cell periphery. Studies of anterior pituitary cells in culture indicate that ACTH-containing secretory granules move to the periphery along microtu buIes (64,1 1 8) (Figure 4). The movement of secretory granules is most likely driven by kinesin. It has been proposed that microtubules are necessary for the correct and efficient packaging of secretory materials into vesicles (41). The morphology of the Golgi apparatus is disrupted by microtubule poisons, but protein processing and transport through the cis, medial, and trans regions of the Golgi are not significantly inhibited. In contrast, the effects of microtubule inhibitors on processing events in the trans-Golgi network such as protein sorting, aggregation, condensation, and packaging into secretory granules are not well understood. The TGN extends tubules and buds off vesicles that allow communication between different regions of the network; motility is believed to be microtubule-dependent (23) (Figure 4). Membrane dynamics may facilitate any of the processes known to occur inthe TGN. Future studies should help clarify the role of microtubules in TGN function. Delivery to Apical and Basolateral Plasma Membrane in Polarized Epithelia

Polarized epithelia have two plasma membrane domains of distinct protein and lipid compositions (see reviews SO, 1 09). The mechanisms for sorting of materials to the two membranes and for maintenance of their distinct compo sitions are currently of great interest. Membrane polarity is commonly studied in three epithelia: kidney (the model system used is the Madin-Darby canine kidney cell line,MDCK),intestine (either intestinal epithelium or the cell line Caco-2), and hepatocytes. Two different mechanisms for delivery of material to the apical membrane have been described: direct transfer from the trans Golgi network to the plasma membrane, and indirect transport wherein materials are first delivered to the basal surface and are then taken up and transported to the apical domain by transcytosis (reviewed in 50). Newly



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synthesized materials destined for the basolateral membrane are transported exclusively by a direct pathway. A central question is the role of microtubules in delivery of membrane and secreted proteins to the apical and basolateral domains. All studies to date indicate that microtubules are not required for delivery to, the basolateral surface, whereas transport to the apical domain by both direct and indirect routes seems to involve microtubule-based motility. In MDCK cells, proteins destined to the different domains are sorted into distinct vesicles as they leave the trans-Golgi network; basolaterally and apically directed materials are transported directly to the appropriate plasma membrane domain ( l 09). In MDCK cells, the role of microtubules in apical delivery remains controversial. In one model the microtubules facilitate the movement of vesicles to the apical membrane. When microtubules are de polymerized, the delivery of membrane proteins, glycolipids, and secreted proteins to the apical surface occurs at a significantly reduced rate, but is not completely inhibited (glycolipids, G. van Meer, K. Simons, personal com munication; membrane proteins, 94). Some reports indicate that inhibition of apical delivery results in mistargeting to the basolateral surface (94), while other reports do not observe mistargeting (in rat kidney; 38). Recent studies by Matlin & co-workers suggest that, in the presence of colchicine, apically directed membrane proteins are degraded at an increased rate (M. van Zeijl, K. Matlin, unpublished results). Others have observed an increase in the intracellular degradation rate of glycoproteins in the presence of colchicine, which suggests that vesicles prevented from reaching the cell surface have an increased probability of being mis-sorted to the lysosomal pathway (13, 96). Since most microtubules in MDCK cells are oriented in longitudinal bundles with minus ends nearest the apical surface (see Figure 2; 9) microtubule-based transport toward the apical membrane would be expected to be driven by cytoplasmic dynein. In other epithelia, components travel indirectly to the apical surface. Hepatocytes use the indirect route exclusively (11). Caco-2 cells have both direct and indirect pathways for transport to the apical domain (76). Microtu bule inhibitors profoundly inhibit apical delivery in both systems (29, 49). The microtubule-dependent step appears to be transport from the basolateral to apical surface (transcytosis), a process that is predicted to utilize a cytoplasmic dynein-based (minus-end-directed) transport mechanism. Endocytic Pathway

The endocytic pathway, like the secretory pathway, involves the movement of vesicular carriers between different membrane compartments; from plasma membrane to endosome to lysosome. For many years investigators have utilized the light microscope to observe the movements of vesicles loaded


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with endocytic tracers. The vesicles move in a saltatory manner and are ultimately translocated into the central region of the cell where lysosomes are found (25, 28, 40, 132, 134). Studies with microtubule inhibitors indicate that uptake of endocytic markers is not microtubule-dependent. However, cen tripetal, saltatory vesicle movements are abolished (12, 25, 40, 126), and degradation of endocytic materials is inhibited (18, 86). This work clearly indicates that endocytic vesicles move centripetally in a microtubule dependent manner in order to deliver their contents to lysosomes. Endosome motility would therefore be directed toward the minus ends of microtubules and should occur via cytoplasmic dynein. In keeping with this prediction, isolated endosomes can be observed to move toward the minus ends of microtubules in vitro (V. Mermall et aI, manuscript in preparation). While the observational approach has allowed the motility of endosomes to be studied in great detail,cell fractionation and immunochemical studies have provided the most information on the biochemical compartments that com prise the endocytic pathway (37). It has recently become possible to correlate the microtubule-dependent events with movement of materials between biochemically defined compartments. In vitro assays that me�w'e-the in termixing and processing of endocytic markers have allpwed the role of microtubules and microtubule-based motors to be tested directly. The first events in the pathway, namely endocytosis, deliverf of markers from the endocytic vesicle into the early endosome, and transfer of markers between early endosomes have been clearly demonstrated to be microtubule independent (26, 27, 37). These assays utilize a crude cell homogenate that contains late endosomes and lysosomes, yet in some cases (26, 27) the endocytic markers were not processed further, which suggested that transfer to later compartments did not occur in vitro. It was suggested that the endosomes age and are not competent to be processed further in vitro. It was also possible that the subsequent steps of movement toward and fusion with late endosomes required microtubules. The elegant study of Gruenberg & co-workers (36) has defined a microtubule-dependent step in the pathway. Endocytic materials move from the early endosome into transport vesicles that mediate delivery to late endosomes and lysosomes. Transfer of materials from the transport vesicles into the late endosome is blocked by microtubule inhibitors, thus suggesting that the process is microtubule-based. Endocytic tracers taken up at the apical and basolateral faces of polarized epithelia are delivered to a common sorting compartment that is effectively a late endosome (14,51, 88). An in vitro assay that measures the intermixing of material taken up from the two domains was developed in MDCK cells and was used to determine the role of microtubules and the microtubule-based motors (kinesin and cytoplasmic dynein) in this process (13a). Both in vivo and in vitro, the key microtubule-dependent step was found to be transfer of


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material. from the early endosome to the common sorting compartment (late endosome) . Removal of either kinesin or dynein led to partial inhibition of transfer. This is consistent with the hypothesis that endocytic vesicles move centripetally along the longitudinal microtubule bundles present in these cells

(9) . According to this model, apically-derived endocytic vesicles should be Annu. Rev. Physiol. 1991.53:629-652. Downloaded from www.annualreviews.org Access provided by University of Texas Southwestern Medical Center on 01/28/15. For personal use only.

subject to kinesin-based motility and basolaterally-derived endocytic vesicles would be expected to be transported by cytoplasmic dynein.

SUMMARY Microtubule-dependent transport is necessary for the intracellular functions of mitosis and axonal transport. In addition, a variety of microtubule-based vesicle movements occur in all cells. Recent studies indicate that the de polymerization of microtubules results in significant inhibition of the recycl ing of the ER membrane from the ER-Golgi transition compartment and inhibition of the maturation of endosomes . Other membrane traffic events are inhibited by microtubule depolymerization, but in most cases alternate path ways can accomplish the function in question . With a more in-depth un derstanding of these alternate pathways and improved tools for inhibiting motor function without affecting cytoplasmic viscosity, we should be able to . determine more precisely the roles of motor-dependent vesicle transport.

ACKNOWLEDGMENTS We would like to thank those investigators who shared their recent studies with us, and we also thank members of the Sheetz laboratory for their helpful comments .

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Catalytic Locomotion of Core-Shell Nanowire Motors.

Molecular motors: Hook-ing up early endosomes.

Molecular motors in the nervous system.

Molecular motors: on track with nanotubes.

Molecular motors: DNA takes control.

Genetic approaches to molecular motors.

Chemistry in motion: tiny synthetic motors.

Roop Mallik: From machines to molecular motors.

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Interferometric Scattering Microscopy for the Study of Molecular Motors.

Methodology for the Simulation of Molecular Motors at Different Scales.

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Molecular motors: Shifting gears with light.