Roles of the cytoskeleton and motor proteins in endocytic sorting

Advanced Drug Delivery Reviews 55 (2003) 1385 – 1403 www.elsevier.com/locate/addr

Roles of the cytoskeleton and motor proteins in endocytic sorting John W. Murray *, Allan W. Wolkoff Marion Bessin Liver Research Center and Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 517 Ullmann Building, 1300 Morris Park Avenue, Bronx, NY 10461, USA Received 10 July 2003; accepted 30 July 2003

Abstract After internalization, endocytic material is actively transported through the cytoplasm, predominantly by microtubule motor proteins. Microtubule-based endocytic transport facilitates sorting of endocytic contents, vesicle fusion and fission, delivery to lysosomes, cytosolic dispersal, as well as nuclear uptake and cytosolic egress of pathogens. Endosomes, like most organelles, move bidirectionally through the cytosol and regulate their cellular location by controlling the activity of motor proteins, and potentially by controlling microtubule and actin polymerization. Control of motor protein activity is manifest by increased microtubule ‘‘run lengths’’, and the binding of motor proteins to organelles can be regulated by motor protein receptors. A mechanistic understanding of how organelles control motor protein activity to allow for endocytic sorting presents an exciting avenue for future research. D 2003 Elsevier B.V. All rights reserved. Keywords: Endocytosis; Microtubules; Actin; Rabs; Kinesin; Dynein; Myosin; Virus; Lipids

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Introduction. . . . . . . . . . . . . . . . . . . . . . . Motor proteins . . . . . . . . . . . . . . . . . . . . . 2.1. Kinesins. . . . . . . . . . . . . . . . . . . . . 2.2. Dyneins . . . . . . . . . . . . . . . . . . . . . 2.3. Myosins. . . . . . . . . . . . . . . . . . . . . 2.4. Conclusion from genome analysis of motor proteins Categorization of cytoskeletal-based motility. . . . . . . . 3.1. Microtubule organization . . . . . . . . . . . . . 3.2. Microtubule plus-end tracking. . . . . . . . . . . 3.3. Actin organization . . . . . . . . . . . . . . . . 3.4. Actin rocketing . . . . . . . . . . . . . . . . .

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* Corresponding author. Tel.: +1-718-430-2584; fax: +1-718-430-8975. E-mail address: [email protected] (J.W. Murray). 0169-409X/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2003.07.008

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How microtubule motors localize endosomes . . . . . . . . . . . . . . . . . . . . . . 4.1. Endosome movement mediated by cytoplasmic dynein. . . . . . . . . . . . . . 4.2. Movement of endosomes and other organelles is bidirectional . . . . . . . . . . 4.3. Endosome motility in ustilago . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Endosome movement mediated by kinesins . . . . . . . . . . . . . . . . . . . 4.5. Conclusions about bidirectional movement . . . . . . . . . . . . . . . . . . . 5. Coordination of bidirectional movement on microtubules . . . . . . . . . . . . . . . . 5.1. Dynein and kinesin II bind dynactin . . . . . . . . . . . . . . . . . . . . . . 5.2. Oppositely moving motors are not in a tug-of-war . . . . . . . . . . . . . . . . 6. Motor protein receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. ‘‘Scaffolds’’ as motor protein receptors . . . . . . . . . . . . . . . . . . . . . 6.2. Delivery of the yeast vacuole to the bud through myo2, Vac8a and Vac17a . . . . 7. Rab proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Rab27a-melanophilin is the receptor for myosin V and pigment granule localization 7.2. Other interactions of Rabs with motor proteins . . . . . . . . . . . . . . . . . 8. Trafficking of viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1. Vaccinia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. Adenovirus type 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3. Other viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Lipids and microtubule-based trafficking . . . . . . . . . . . . . . . . . . . . . . . . 9.1. PIP2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2. Cholesterol and Niemann-Pick C disease . . . . . . . . . . . . . . . . . . . . 10. Conclusions and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1. Domain displacement overexpression . . . . . . . . . . . . . . . . . . . . . 10.2. Drug delivery and disease . . . . . . . . . . . . . . . . . . . . . . . . . . 10.3. Drugs that inhibit specific motor proteins . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Recent studies have revealed an important role of microtubules and microtubule motors in endocytic trafficking. New experimental techniques and a greater knowledge base are catalyzing the scientific exploration of intracellular vesicular trafficking. In this review, we will focus particularly on how the measurement of microtubule and actin based movement is contributing to our understanding of endocytic processing. During receptor-mediated endocytosis, ligands bind to specific cell surface receptors and are internalized within clathrin-coated pits whose constriction leads to coated vesicle formation. Clathrin is released rapidly and uncoated vesicles mature into early/sorting endosomes [1 –3]. Acidification of early endosomes [4] leads to dissociation of most receptor – ligand complexes [5,6]. Early endocytic vesicles undergo a series of fusions and fissions resulting in the segregation of receptor from ligand. Ligands destined for degradation traffic within late endo-

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somes to lysosomes while receptor-enriched vesicles are recycled back to the cell surface [1,2,7,8]. Microtubules are dynamic protein filaments that stretch across cells and provide a mechanical basis for chromosome sorting, cell polarity and organelle localization among other functions. In vivo, microtubules grow and shrink from the ‘‘plus-ends’’, whereas the ‘‘minus-ends’’ of the microtubules are usually located at the microtubule-organizing center as well as the apical domain in epithelial cells and the axonal terminus in neurons. The transport of endocytic contents from early to late endosomes is dependent on microtubules and microtubules can promote fusion and fission of endocytic vesicles [7,9 – 12]. Direct observation of endocytosis by microscopy shows that endocytic processing occurs in association with microtubules [8,13,14]. Filamentous actin drives muscle contraction, cytokinesis and extension of the plasma membrane, and can form branched, isotropic intracellular networks and well-organized bundles. It can also participate in endocytic processing events of clathrin-mediated

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uptake and sorting from early to late endosomes [15]. Actin filaments have a ‘‘barbed-end’’ where filament growth tends to occur, and the barbed-ends are abundant in the cortex of the cell facing the membrane. Endocytic traffic has been shown to occur through endocytic carrier vesicles and this has been fueled through study of synaptic vesicles [16]. Carrier vesicles have been shown to shuttle between different organelles using an assortment of recognition and fusion-promoting proteins (e.g. v-SNAREs, tSNAREs). However, the physiological strategy and energetics of these events are not easy to understand. Membrane trafficking events are complex and involve thousands of different kinds of proteins. The obligatory localization of these proteins through space and time might be expected to create organizational problems: complicated machines break easily. But intracellular traffic does not break easily. Instead, evolution has produced endocytic machinery that is both complicated and robust.

2. Motor proteins A recent analysis of the distribution of motor proteins across eight eukaryotic species has distilled the large array (humans have 45 kinesin, 15 dynein and 40 myosin genes) of known motor proteins into 5 kinesin, 3 dynein and 2 myosin subclasses based on comparison of sequences and known functions of the motors [17]. Two fifths of the myosin and kinesin genes did not fall into any subclass, and other classification schemes may provide additional functional insight. However, this analysis provides a useful, simple hypothesis for understanding motor proteins. The classes can be thought of as ‘‘tool box’’ proteins as they represent the putative types of motors that were available to ancient organisms. 2.1. Kinesins Kinesins bind microtubules and hydrolyze ATP to produce movement toward the fast-growing, plus-ends, of microtubules. ‘‘C-terminal’’ kinesins, which have their motor domains at the C-terminus of the protein, move in the opposite direction, toward the minus-ends. Of the five proposed subclasses of kinesins, conven-

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tional kinesin, kinesin II and Unc104 can transport cargo, while members of the C-terminal subclass may also provide this function (see below). Mitotic kinesins, as the name implies, appear to function predominantly in mitosis. Kinesins generally exist in the iconic motor protein structure of an elongated dimer with an ATPhydrolyzing motor domain at the N-terminus followed by a central coiled coil region, where the dimers wind together, and ending in a C-terminal, light chain binding tail region. The tail region, either itself or through light chains, is frequently the site of cargo interaction. The ‘‘cargo’’, being the vesicle, protein, RNA, or other substance that is dragged through the cell along microtubules. This design permits separate regulation of the ATP-hydrolysis and cargo-binding functions. Conventional kinesins (the KIF5s) have been shown to transport mitochondria, lysosomes, endoplasmic reticulum, mRNA and nerve axon organelles [18]. Kinesin II proteins (KIF3s, KIF17) transport cargo to flagella or cilia and power intraflagellar transport, the specific movement of vesicles through flagella [19]. Kinesin II can also move pigment granules and neuronal vesicles [20]. Fungi, which do not have flagella, also do not have kinesin II proteins, and a kinesin II knockout mouse has ciliary defects and dies during embryonic development [21]. A kidney specific knockout of kinesin II results in polycystic kidney disease [22]. Unc104 proteins (KIF1s, KIF13s, KIF14, KIF16s) are responsible for cytosolic vesicle transport in Dictyostelium and axonal transport in the nematode and vertebrates [23]. Mitotic kinesins are the only subclass of motor proteins present in every eukaryote so far sequenced. These kinesins allow chromosome segregation and deletions are often lethal [24,25]. Inhibitors of these kinesins are currently being developed as anti-tumor agents [26]. C-terminal kinesins appear to be involved in transport of neuronal multivesicular bodies and Golgi. Our laboratory has reported that KIFC2 is present on hepatocyte endosomes and an antibody to KIFC2 blocked minus-end movement of these vesicles in vitro [27]. Ncd, the first minus-end directed kinesin discovered, participates in mitosis and chromosome segregation in Drosophila [28]. Interestingly, deletion of either KIFC2 or KIFC3 in mice produced no obvious phenotype [29,30].

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Despite the association of microtubule directionality with the C- or N-terminal motor domain location, the mechanical basis for directionality is still unclear. Mutations in the ‘neck linker’ region adjacent to the motor domains can reverse the direction of movement [31]. Although motors that move toward both minus- and plus-ends of microtubules have not been found in nature, they can be created in the laboratory [32]. 2.2. Dyneins Dyneins are large (1 –2 MDa) microtubule motors with very different structures from the much smaller kinesins. The motor domain is composed of a hexamer of AAA domains (ATPases Associated with cell Activities). These domains are present in a wide variety of proteins and appear to bind and release proteins in an ATP-dependent manner [33]. Recent electron microscopy studies beautifully reveal the ‘‘pinwheel with arms’’ structure of dynein where, as opposed to kinesins or mysosins, the head domain is centrally located and apparently rotates causing the microtubule-binding arm to move in a 15-nm ‘‘power stroke’’ [34]. Dynein motors have been assigned to three subclasses: cytoplasmic, intraflagellar transporting and axonemal. Almost all of the human genes are axonemal and presumably drive ciliary and flagellar beating. Cytoplasmic dynein, which drives a huge array of organelle transport, has only one heavy chain gene in most organisms. Dynein makes up for its lack of gene diversity with a large array of associated proteins including light intermediate chains, intermediate chains and several types of light chains that appear to facilitate specific cellular localization of dynein. In addition, the important multi-subunit complex, dynactin, binds dynein and stimulates long distance movement of dynein. Overexpression of the dynamitin (p50) subunit of dynactin disrupts the dynactin complex and inhibits dynein-driven events of mitosis and organelle transport [35 – 37]. 2.3. Myosins Myosins make up the actin-based motor family and structurally resemble kinesins with a globular motor domain at the N-terminus followed by an

extended coiled-coil, and finally a globular tail domain that interacts with cargo proteins. Myosin and kinesin may actually share a common ancestor [38]. The large variety of myosins have been placed into just two subclasses [17]: the class II musclecontraction class, which can polymerize to form long structures with actin such as the sarcomere (muscle contraction) and contractile ring (cytokinesis), and the myosin V (five) class, which can drive vesicle transport. Myosins move toward the barbedends of actin where filament growth occurs. This is analogous to kinesin’s movement toward the plusends of microtubules. But, again analogous to kinesin, there are rogue myosins (e.g. myosin VI) that move towards the slow growing, pointed-ends of F-actin. 2.4. Conclusions from genome analysis of motor proteins Different species apparently use different types of motors for similar tasks. Plants as well as yeast (S. cerevisiae) appear to use actin for organelle traffic. Arabidopsis has no dyneins, though other plants do have dyneins, and no conventional cargotransporting kinesins. Instead, Arabidopsis has an abundance [13] of myosin V genes and a super abundance [21] of C-terminal kinesins. Multicellular organisms tend to have more motor protein genes, but not always. Giardia (a protozoan) has 25 kinesins and 10 dyneins, while C. elegans (a metazoan) has 20 kinesins and only 2 dyneins. One could speculate that additional motor protein genes would have evolved to carry particular cargo. However, humans, like most species, have only one cytoplasmic dynein gene and only three myosin V genes. These proteins transport many kinds of cargo, so specificity for particular cargo would have to come from somewhere else.

3. Categorization of cytoskeletal-based motility The geometry of the cytoskeleton appears to allow two types of movement in mammalian cells: short, non-directed movement on actin filaments and long, directed movement on microtubules. However, the cytoskeleton architecture varies greatly between cell

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types and this represents a major defining factor for differentiated cells. 3.1. Microtubule organization In the interphase hepatocyte, microtubules emanate from the apical domain (the bile canaliculus) and centriole to form an umbrella-like cage [39,40]. Vesicles could use minus-end directed microtubule motors to reach either the bile or centriolar regions. Plus-end directed microtubule motors could deliver cargo to the periphery or basal-lateral membrane. Actin in the hepatocyte is concentrated in the cortex near the periphery and especially adjacent to the bile canaliculi. Non-bundled actin filaments at cell peripheries generally form branched or dendritic arrays composed of short, labile filaments [41,42]. Because the filaments are not polarized in a single direction, myosin is unlikely to transport vesicles in well-directed fashion in these cells. In neurons, cells of extreme polarity, bundles of microtubules stretch from the cell body to the tips of the axon where the plus-ends are localized, sometimes more than a meter away. Axonal transport of organelles, RNA and protein along the microtubules is absolutely required for health of neurons, and a growing list of human diseases potentially have their bases in faulty or blocked axonal transport [43]. Myosin V can move organelles in neurons but microtubules are primarily responsible for rapid long distance movement. In neurons lacking myosin V, organelles become stuck in axon terminations and branch points, suggesting that myosin V is responsible for movement or redistribution out of these relatively wide regions [44]. 3.2. Microtubule plus-end tracking Proteins that attach to growing ends of microtubules form another type of long-distance microtubule-based movement. CLIP-170, EB1 and dynactin are so called ‘‘plus-end tracking proteins’’ that attach to microtubules while they grow and thereby become transported to distal parts of the cell. It appears that these proteins regulate microtubule dynamics. The back-and-forth growth of microtubules to the cell cortex constitutes a probing activity for such things as cell – cell contact, polarized growth and mitotic spindle pole localization. CLIP-170 was initially iso-

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lated as a ‘‘Cytoplasmic LInker Protein’’ thought to link endosomes to microtubules. However, it is not yet clear how this plus-end tracking activity relates to organelle movement and distribution [45,46]. 3.3. Actin organization There are many types of actin bundles with different amounts of order. Filopodia, the thin fiber projections at the membrane of cells, and microvilli, the hair-like extensions in epithelia, contain actin bundles of uniform polarity with barbed ends facing outward. An in vitro system has been used to form such bundles and the uniform polarity appears to rely on remodeling of existing actin filaments [41]. Motor proteins can utilize parallel actin bundles as demonstrated by elegant experiments on the function of myosin-1c in the adaptation response in hearing. Myosin-1c most likely works by walking up the parallel actin bundles in the stereocilia of inner ear hair cells, generating membrane tension that adjusts the conductance of tension-gated ion channels [47]. It seems that such long unipolar bundles are not generally present in cells, however. Studies of fibroblasts and the epithelial cell line, PTK2, showed that stress fibers, the large actin bundles seen in many tissue cultured cells, have sarcomere-like bipolarity; short stretches of bundled, unipolar filaments are thatched against each other along the stress fiber axis [48]. Another arrangement is the ‘‘mixed graded polarity’’ bundle [49]. In these bundles, actin filaments have their barbed-ends out near the cell periphery but have mixed polarity in the middle of the cell. It is unlikely that myosins could make net progress along stress fibers, but they could make net progress towards the cell periphery on graded polarity bundles using repeated cycles of movement and detachment. Myosin VI moves toward the pointed-ends of actin filaments and is found at the leading edges of cells where pointed-ends of filaments face inward. Because of this geometry, myosin VI could pull plasma membrane inward. Indeed, myosin VI appears to associate with clathrin and can bind proteins that interact with cell surface receptors. Deletions in yeast and Dictyostelium myosin VI result in endocytic defects [50,51]. Surprisingly, endocytic granules in astrocytes move in a directed but episodic nature towards the cell center apparently utilizing myosin V. Overexpres-

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sion of myosin tail domains lacking the myosin motor (dominant negative constructs) as well as treatment with myosin inhibitors block this movement whereas microtubule depolymerizing drugs do not [52]. As discussed, most actin filaments and actin bundles are not expected to have their barbed-ends towards the nucleus so the mechanism by which myosin V could be moving the vesicles is not clear. Myosin V distributes pigment granules in melanocytes (called ‘‘melanophores’’ in reptiles and amphibians), as has been shown in many species [53 –55]. In Xenopus melanophores, dispersal and aggregation of pigment granules is achieved through myosin V, kinesin II and cytoplasmic dynein. Myosin V appears to compete with the microtubule-based motors by pulling the granules off microtubule tracks and on to the more randomly oriented short actin tracks. Elimination of myosin V activity through dominant negative constructs causes aggregation of the pigment granules at the cell center and also increases the number of stalled granules. Myosin V appears to provide constant movement and distribution of the granules, which is critical for their eventual localization [56]. 3.4. Actin rocketing Actin rocketing is another form of actin-based motility used by organelles. First discovered as a means of propulsion for parasitic bacteria [57], it has since been detailed that actin polymerization at the surface of intracellular objects will drive their movement through the cell. Movement results from incorporation of actin subunits at the surface, which can steadily push the object at f 0.1– 0.5 Am/s [58]. This same motility mechanism has been shown to push pinosome vesicles away from the cell membrane [59], indicating that organelles may use actin polymerization based motility, which does not require motor proteins, for some motile events. Actin rocketing appears analogous to microtubule plus-end tracking, and the potential parallels may indicate the kind of protein polymer physics that is available to cells.

4. How microtubule motors localize endosomes Kinesins and dyneins move in only one direction along microtubules. Dyneins move toward the minus-

ends and kinesins (except for the C-terminal variety) move toward the plus-ends. Therefore, membrane traffic on microtubules should follow a simple rule: traffic destined for the cell surface should move by kinesins, traffic destined for either the cell center or apical domains should use cytoplasmic dynein. 4.1. Endosome movement mediated by cytoplasmic dynein Endocytic trafficking, which proceeds from the cell surface and terminates at perinuclear lysosomes, presumably involves cytoplasmic dynein. Indeed dynein is found associated with late endocytic vesicles and lysosomes in liver [60,61] and dynein and its activator, dynactin, are found on early endosomes in cultured macrophages [62]. Dynein can stimulate early-to-late endosome vesicle fusion [11], and dominant-negative overexpression of the dynactin subunit, dynamitin, inhibits endocytic processing, stops endocytic movement and blocks traffic to lysosomes [63]. An antidynein monoclonal antibody (mAb anti-IC74, Chemicon International) has been shown to inhibit endocytic transport in several systems [64,65]. One caveat to these studies is that the inhibition of dynein may also inhibit other motors, and dynactin was recently shown to also bind kinesin II (see below) [66]. However, the studies clearly indicate that dynein is involved with endosome traffic. 4.2. Movement of endosomes and other organelles is bidirectional Direct observation of the movement of endosomes demonstrates a complicated situation [13,67 – 69]. Endosomes move bidirectionally in the peripheral regions of cells sometimes followed by uniform centripetal movement. Their eventual juxta-nuclear localization is established through small biases in minus-end movement as well as retention or anchoring of the endosomes near the nucleus. A broader perspective shows that most organelles move bidirectionally and may switch between the plus and minus directions on the time scale of once a minute [70]. Organelles can move unidirectionally in axons and flagella where the organelles must traverse long distances towards a specific distal destination (the axon or flagella terminal). For organelles such as endo-

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somes, Golgi, endoplasmic reticulum and mitochondria, movement tends to be episodic, i.e. back and forth. This means that both minus-end directed motors (either dynein or C-terminal kinesins) and plus-end motors (kinesins) attach to these organelles. 4.3. Endosome motility in ustilago Interesting studies in the corn fungus, Ustilago Maydis, showed that knocking out a Unc104 (KIF1a) kinesin reduced the number of moving endosomes by 67%, and all endosome motility ceased when dynein function was also disrupted by use of a mutant dynein heavy chain allele. Endosome cluster location appears to be achieved by regulation of the ‘‘run lengths’’ of the traveling endosomes. A run length is the distance traveled by an individual endosome before it pauses or falls off a microtubule. Accumulation of endosomes at the minus-ends of microtubules (e.g. the emerging bud) corresponded to increased minus-end run lengths, whereas accumulation of endosomes at plus-ends of microtubules corresponded to increased plus-end run lengths. Overexpression of the KIF1A kinesin increased plus-end and decreased minus-end run lengths [71]. 4.4. Endosome movement mediated by kinesins Other studies show that kinesins are important for endocytic processing. The movement of early endosomes from hepatocytes on microtubules was dynein-independent as seen by in vitro assays [8,72] and the bidirectional nature of this movement appears to depend on plus- and minus-end directed kinesins. The minus-end kinesin, KIFC2, was detected on these endosomes. KIFC3, another minus-end directed kinesin, has been shown to affect Golgi distribution when cholesterol was depleted [68], a condition that removes dynein from endosomes [73]. Microinjection of a kinesin monoclonal antibody inhibited transferring recycling [74,75], and kinesin is detected in purified endosome fractions [76]. 4.5. Conclusions about bidirectional movement We conclude then that the steady state localization of most organelles is achieved by the regulation of

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minus- and plus-end microtubule-based movement, along with possible anchoring of the organelle, and that altered microtubule ‘‘run lengths’’ may represent the most common manifestation of the regulation of direction. The use of both plus- and minus-end directed motors would seem to have the logical advantage that the localization of organelles could be modified and fine-tuned. It has also been proposed [8,60,69] that the bidirectional movement of endosomes provides a means for cells to physically segregate vesicle contents.

5. Coordination of bidirectional movement on microtubules 5.1. Dynein and kinesin II bind dynactin Recently, several studies have been published that address the coordination of opposite-directed motors in organelle movement [20,66]. Dynactin, a protein known to link cytoplasmic dynein to organelles as well as stimulate minus-end cytoplasmic dynein movement, has been shown to specifically bind the KAP protein of heterotrimeric Kinesin II, a plus-end directed microtubule motor, and the protein-binding region has been mapped. Several studies had previously shown that inhibition of dynactin blocked both plus- and minus-end movement of organelles and the result was difficult to explain since dynactin was known to bind only dynein [77,78]. Inhibition of kinesins also has led to loss of both plus and minusend directed movement [79,80], and interactions of kinesins with dynactin has been observed [81]. The new finding shows that KAP and Dynein intermediate chain cannot bind dynactin p150 at the same time, suggesting a mechanism by which plus and minus direction can be coordinated: kinesin and dynein motors displace each other at the vesicle. 5.2. Oppositely moving motors are not in a tug-of-war Despite the findings with KAP and dynein intermediate chain, it is known that dynein and kinesins can bind to the same vesicle simultaneously. How does the vesicle decide to move one way versus the other? One possibility that has been considered is that the motors pull in opposite directions, and the winner

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of the tug-of-war moves the vesicle. One in vitro analysis suggested that kinesins pull harder than dynein [82]. Recently, Gross et al. [66] studied the bidirectional movement of Drosophila lipid droplets and found that mutations in dynein that mildly decreased minus-end movement and pulling force resulted in decreased plus-end movement and force. They also found that a dynactin p150Glued mutant reduced minus-end run lengths and reduced plus-end run lengths and increased plus-end pausing. These in vivo results don’t fit with a tug-of-war scenario. The authors instead propose that motors turn off during oppositely directed movement. Unfortunately, the amount of dynein and kinesin bound to the vesicles was not measured in these studies. It might be possible for motor ‘‘displacement’’ to function as the turn off mechanism, i.e. dynein protein could displace kinesin on the surface of the vesicle during minus-end directed movement and, subsequently, kinesin could displace dynein during plus-end movement. However, other studies have reproduced bidirectional motility in vitro in the absence of soluble motor proteins [67], a situation where this hypothetical motor displacement is unlikely to occur. Myosin V and kinesin heavy chain have also been shown to interact directly, and vesicles can switch rapidly between microtubule and actin tracks, suggesting that the activity of these motors also must be coordinated [83].

6. Motor protein receptors Motor proteins appear to have ‘‘receptors’’ that link them to their cargo. This could provide the mechanism by which motors find specific cargo within the cell. It is not absolutely clear that motor proteins require specific protein receptors; they generally are active on glass or latex beads, and they are able to bind directly to lipids under some conditions [84 – 86]. One group has suggested that motors may need receptors in order to distribute their pulling force over a large surface area so that lipid molecules are not simply pulled off [87]. 6.1. ‘‘Scaffolds’’ as motor protein receptors Many specific motor protein receptors have been discovered and the list is growing rapidly [88]. Al-

though in most cases we do not understand the physiological strategies for the regulated attachment of motor proteins to membranes and vesicles, some generalizations have emerged. Many motor protein receptors are associated with large protein complexes or coats bound to vesicles. Fodrin (non-erythrocyte spectrin), which forms long filamentous polymers across cell membranes and binds actin and actinbinding proteins, appears to be a receptor for KIF3 (kinesin II) in fast-axon transport vesicles [89]. Fodrin also appears to link cytoplasmic dynein and dynactin to Golgi, or protein free liposomes [90,91]. The KAP subunit of kinesin II binds dynactin, while both KAP and dynactin also bind fodrin [92]. Conventional kinesin has been shown to bind the JNK kinase scaffolding proteins, the JIPs (c-Jun Nterminal kinase Interacting Proteins). A mutation that causes aberrant axonal transport in Drosophila [93] was found to be in a JIP3 homologue, and similar effects were subsequently seen in C. elegans [94]. Another group found that the kinesin-JIP interaction occurs through tetratricopeptide repeats of the kinesin light chain [95]. Scaffolding proteins are proposed to tether kinases to specific membrane locations [96]. The paradigm of a scaffold protein came from the yeast Ste5p, which was found to bind multiple members of the MAP kinase cascade [97]. Scaffolding proteins are thought to oligomerize and form multiprotein matrices and, in so doing, regulate protein function, especially kinases. In fact, enzyme tethering may become a valuable tool for artificial protein design [98]. Multiple light chain subunits can bind to the stem region of dynein. These light chains can provide specific cargo attachment sites to proteins to which they bind. Light intermediate chain isoform 1 and isoform 2 bind to the same site on dynein heavy chain but only isoform 1 binds the centrosomal protein, pericentrin. Therefore, light intermediate chain 1 provides a means to attach and transport pericentrin through dynein [99]. Tctex-1, another dynein light chain, so named because its gene exists in the chromosomal mouse t-complex associated with mitotic drive and male sterility [100], has been found to bind to the visual pigment, rhodopsin. In MDCK cells overexpression of a similar but non-rhodopsin binding dynein light chain, RP3, reduces the amount of cellular tctex-1 and disrupts the proper apical targeting

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of rhodopsin [101]. Transport of flagellar cargo may also be achieved through a specific dynein light intermediate chain [102]. 6.2. Delivery of the yeast vacuole to the bud through myo2, Vac8a and Vac17a Studies on the yeast myosin V (myo2) protein demonstrate how cargo may be selectively transported. As mentioned, S. cerevisiae and plants tend to use actin for vesicle transport through cells. The C-terminal tail domain of myo2 is responsible for binding secretory vesicles and delivering them to the growing bud [103]. One region of the tail appears to target to the vacuole while another region, separated by about 150 amino acids, appears to target to sites of bud growth (vacuolar inheritance) [104]. Myo2 attaches to vacuoles in the mother by binding to Vac17p, the myo2 receptor, which is bound to Vac8p, a resident vacuolar protein. Myo2 crawls into the bud with the vacuole attached through Vac8p– Vac17p. In the bud, Vac17p is specifically degraded. This allows myo2 to detach, thus delivering the vacuole. This likely represents the first well-characterized example for understanding the complete ‘‘delivery pathway’’ of any cargo and appears to indicate a physiological strategy used by cells. Whether such a strategy may be generalized to different organisms remains to be seen.

7. Rab proteins The Rab G-protein family forms another group of potential receptors for motor proteins. There are about 60 Rab genes in humans and they are involved in many intracellular trafficking events and localize to specific intracellular domains [105]. Rabs appear to link proteins to membranes in a GTP-dependent manner and, in this way, they may act as receptors for motor proteins. Rabs may bind motor proteins or their subunits directly or through intermediary proteins. 7.1. Rab27a-melanophilin is the receptor for myosin V and pigment granule localization Myosin V interacts with Rab27a apparently through the protein melanophilin. Ashen, dilute

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and leaden are mouse mutant alleles giving rise to washed out coat color. The genes of these code for Rab27a, Myosin Va and melanophilin, respectively, and the melanocytes from these mice have mislocalized pigment granules (melanosomes). In the mutants, melanosomes concentrate near the nucleus instead of at the periphery and this prevents adequate pigment uptake into hair. Melanocytes with mutations in either Rab27a or myosin Va have normal microtubule-based motility but cannot capture melanosomes in the periphery. Rab27a appears to link melanophilin, which links myosin Va to the melanophore membranes. Expression of the N-terminal and C-terminal portions of melanophilin induces melanosome nuclear accumulation presumably by displacing the endogenous molecules. Further, it was shown that a specific intron of myosin Va was required for the melanosome targeting [106 – 108]. 7.2. Other interactions of Rabs with motor proteins Expression of a protein, RILP, that binds Rab7 by yeast two hybrid analysis has been reported to recruit dynein and cluster late endosomes – lysosomes near the nucleus [109,110]. Expression of the C-terminal domain of RILP protein disperses the late endosome – lysosomes. This protein may link cytoplasmic dynein to Rab7 in a similar way as melanophilin links myosin Va to Rab27a. However, the endogenous protein has not been examined and some of the effects of this protein are unexpected, i.e. it is not clear how expression of this protein can overcome a dominant negative Rab7 mutant, which normally blocks late endosome to lysosome segregation. Also, by yeast two-hybrid analysis, dynein light intermediate chain-1 was found to interact with Rab4 [111]. Overexpression of these proteins found them co-localized near the nucleus of Hela cells. Recently, our laboratory has discovered that Rab4 may have a somewhat different role in early endosomes from rat liver [27]. In these in vitro experiments, Rab4 was found to co-localize with endosomes, and GTP-g-S Rab4, but not GDP – Rab4, inhibited endosome motility. GDP alone was found to stimulate minus-end directed motility of these vesicles, which were associated with the minus-end directed kine-

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sin, KIFC2, but not with dynein. We hypothesize that GTP-Rab4 inhibits KIFC2 and that GTP hydrolysis on Rab4 allows segregation of endocytic vesicles by increasing minus-end motor activity. Other studies have found that Rab5, which is found associated with early endosomes in many studies, increases early endosome motility, and anti-kinesin antibodies inhibited this motility [112]. These experiments suggest that Rabs may not only function to allow binding of motor molecules to vesicles, but they may regulate the activity of the motor proteins. By two-hybrid analysis Rab6-kinesin (RB6K or KIF20) was found to interact with GTP-Rab6 [113]. However, endogenous RB6K concentrates at the mitotic cell mid-body and overexpression or microinjection of antibodies lead to defects of mitosis. Rab6-kinesin is also related to the mitotic kinesin, MKLP-1 [114,115]. In Drosophila, Rab6 was shown to interact with Bicaudal-D, an oocyte determination factor that can bind dynactin. Overexpression of a truncated Bicaudal-D that cannot bind dynactin leads to a dispersal of the Rab6-positive vesicles [116].

8. Trafficking of viruses Exploitation of the host cytoskeleton appears to be a universal characteristic of pathogen invasion of a host cell. An exciting possibility is that the pathogen-induced cytoskeletal reactions might represent targets for the development of preventative or therapeutic strategies against infection. Pathogen takeover of cytoskeletal machinery certainly appears critical for many kinds of infectivity and cell death [117 –119]. Studies of cytoskeletal-pathogen interactions have already yielded important information on the basic biology of both the cytoskeleton and pathogen. For instance, studies of the intracellular pathogen Listeria monocytogenes have been instrumental in developing quantitative models for the in vivo, multiprotein process of actin polymerization [57,58,120,121]. Listeria and other pathogens use actin rocketing to propel themselves through the cytoplasm and into adjacent cells. The intracellular growth of Salmonella initially involves formation of an actin network

around early Salmonella vacuoles, and this is followed by the fusion of late endocytic compartments with the vacuoles along microtubules to form Salmonella-induced filament structures [122,123]. 8.1. Vaccinia An interesting set of studies describes the microtubule- and actin-dependent trafficking of vaccinia virus [124 – 126]. Vaccinia is a large DNA virus related to smallpox that is used in smallpox vaccines. During infection, vaccinia fuses with the plasma membrane of the target cell and makes its way to the cell center, where it can enter the nucleus. Upon maturation, the virus finds its way to the cell periphery, where it can exit and infect other cells. The exiting virions exhibit both slow, steady peripheral movement and rapid episodic movement. The episodic movement is microtubule-based (maximum average velocity of 0.8 Am/s, bidirectional), while the processive movement is due to actin rocketing (0.2 Am/s, unidirectional). An integral membrane protein, A36R, was found to allow actin rocketing, but this required the phosphorylation of two tyrosine residues. Vaccinia lacking A36R protein failed to show microtubule or actin motility, but substituting the tyrosines with phenylalanine (preventing phosphorylation) allowed microtubule movement but not actin rocketing. These data suggest that transport on microtubules allows mature vaccinia to reach the cell periphery, where the virions are then pushed out and into adjacent cells by actin rocketing. 8.2. Adenovirus type 2 Careful documentation of the movement of adenovirus type 2 showed that virus particles were endocytosed by cultured cells, escaped the endosome after 30– 60 min and made their way to the perinuclear microtubule-organizing center where they could enter the nucleus to continue their life cycle. The motility of these ‘‘naked’’ (non-endosomal) viruses was rapid (up to 2.6 Am/s) and characteristically bidirectional, with periods of net progress followed by pauses and periods of rapid direction switching. Mutant virus that could not escape the endosome also showed microtubule-based motility but did not concentrate near the nucleus. Overex-

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pression of dynamitin decreased minus-end movement and increased plus-end movement and led to a random distribution of the naked viruses, suggesting the movement was partially due to dynein [70].

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differentiated phenotypes may be an important criterion for viral activity.

9. Lipids and microtubule-based trafficking 8.3. Other viruses Another study highlights the fact that similar viruses may use different methods of infection and intracellular transport [127]. Infection of SVG-A cells (human glial cell line) by the human polyoma virus, JCV, was blocked by ammonium chloride, cytochalasin D, acrylamide and nocodazole, while SV40 infection was only blocked by nocodazole. Overexpression of dynamitin did not block infection by either virus. The P protein of Lyssavirus, which causes rabies, was shown to bind to the dynein light chain, LC8, by yeast two-hybrid analysis, co-immunoprecipitation and immunofluorescent co-localization [128,129]. Minus-end directed transport of the ribonuclear virus seems essential, as the virus must travel from the sites of infection (e.g. a peripheral animal bite) to the central nervous system, and nerve ligation can prevent infection. HIV reverse transcriptase particles have also been observed trafficking towards the nucleus upon infection, and retroviral gag proteins appear to interact with the kinesin, KIF4 [64,130]. As seen from these examples, many studies have now described the intracellular trafficking of pathogens, particularly viruses, through mammalian cell cytosol [131]. Microtubule trafficking can occur for enveloped as well as ‘‘naked’’ virus particles, and the microtubule-based movement appears characteristic and bidirectional as is seen for organelle movement. Drugs that affect cytoskeletal arrangement may affect trafficking of similar viruses in different ways. These studies represent a new way to categorize viral infection; additional investigation will be required to assess how motility parameters relate to pathogen biology and disease pathogenesis. More work also is necessary to determine the molecular strategies used by the viruses. What proteins are used and how do they recruit the necessary motor protein machinery to allow microtubule trafficking? It is important to remember that the relevant viral targets for pathogenesis exhibit characteristic differentiated cytoskeletal architecture. Trafficking within cells displaying

Membrane traffic by definition involves transport of lipids (e.g. triglycerides, phospholipids, sterols) through the cell. Some interesting concepts have emerged concerning the possible structural or regulatory role of specific lipids in endocytic traffic. 9.1. PIP2 Phosphatidyl inositol (4,5) bisphosphate (PIP2) is a low-abundance phospholipid that is created from the fairly abundant phosphatidyl inositol (2– 8% of the phospholipids in mammalian cells) by the action of kinases and phosphatases. The binding of G-proteinlinked receptors leads to cleavage of PIP2, yielding inositol trisphosphate and diacylglycerol that can increase calcium and protein kinase C activity. It also now is clear that many proteins specifically bind to PIP2 or one of the other phosphatidyl inositides. Plextrin homology (PH), FYVE and epsin N-terminal homology (ENTH) sequences on proteins can all allow binding to phosphatidyl inositols. PIP2 seems to cluster on the inner leaflet of cell membranes and nucleate cytoskeletal and other matrix formations. Clathrin lattices form around these domains as AP-2 (through mu2), epsin and other proteins bind PIP2 [132,133]. The vesicle trafficking motor, Unc104 (KIF1a), has a PH domain and can directly interact with PIP2 on protein free liposomes and move these liposomes along microtubules. Movement of the liposomes is dependent on high concentrations of PIP2 or conditions that are reported to cluster PIP2 (addition of the lipid ganglioside GM1 and cholera toxin, cholesterol and sphingomyelin, or spermine to the membranes) [84]. These data suggest that the formation of lipid clusters, or ‘‘rafts’’, containing PIP2 could activate KIF1a to pull vesicles or tubules from an organelle or plasma membrane. Non-erythrocyte spectrin has a PH domain and can link cytoplasmic dynein [90] and KIF3 to membranes, illustrating that motor clustering by this method may not be limited to KIF1a. Other reports have shown that hydrolysis

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of PIP2 or methods that reduce PH-PIP2 interactions lead to a lowering of cytoskeletal membrane attachments [134].

lipids and time-lapse microscopy may lead to important insights regarding subcellular lipid trafficking.

9.2. Cholesterol and Niemann-Pick C disease

10. Conclusions and future directions

Another line of vesicle trafficking studies demonstrates that cholesterol is important for endocytic vesicle processing. Niemann-Pick type C disease is a rare, fatal, recessive, neurodegenerative disorder predominantly caused by mutations in the integral membrane protein, NPC1. Cholesterol is normally found at the plasma membrane and in early endosomes and is sorted away from late endosomes and lysosomes. In Niemann-Pick C disease, cholesterol accumulates in late-endocytic vesicles, and these lack their normal bidirectional microtubule-based motility. Fluorescently tagged, wild-type NPC1 cholesterolcontaining organelles move throughout the cell with back-and-forth tubulating behavior that appears to allow cholesterol sorting. In Niemann-Pick C mutant cells or in cells overloaded with cholesterol, this microtubule-based sorting behavior is blocked. One hypothesis is that mis-sorting of cholesterol due to dysfunctional NPC1 leads to inhibition of normal vesicle trafficking to axons and results in neurodegeneration [135– 137]. Other studies have shown that cholesterol accumulation in late endocytic compartments leads to increased dynein activity over kinesin: cholesterol overload increases dynein activity, whereas cholesterol depletion reduces dynein activity [138]. Rab7 may be involved in the recruitment or activation of dynein since Rab7 localizes to the cholesterol sorting compartment and overexpression of Rab7 produces a similar cellular phenotype as produced by cholesterol overload [73]. Lipid concentrations are asymmetric throughout endocytic and other trafficking pathways, yet the influence of specific lipids on membrane trafficking events are not well understood [139]. An interesting chicken-or-egg question is whether lipids control the vesicular mediated traffic between organelles or whether trafficking of the organelles controls the lipid distribution. One set of experiments showed that artificial lipids with long-chain, saturated fatty acids sort to late endosomes while short-chain, unsaturated analogues remain in early or recycling compartments [140]. Future experiments with fluorescently tagged

In this chapter, we have discussed recent research concerning motor-dependent vesicular traffic especially as it relates to endocytic sorting. Fig. 1 summarizes much of the recent information on cytoskeletal-driven endocytic sorting events. In this figure, we categorize endocytic sorting into early endosome fusion (EE fusion), early endosome fission (EE fission) and late endosome fusion (LE fusion), as these are topological rearrangements observable by microscopy. These designations correspond roughly to the compartment of uncoupling, the recycling compartment and the multivesicular body compartment, respectively. References that specifically address motor protein and cytoskeleton interactions with endocytic processing have been included. Viral trafficking, myosin V motility, plusend tracking (CLIP-170) and actin rocketing have been included to illustrate additional roles of the cytoskeleton in endocytic processing. Organelles tend to move bidirectionally on microtubules, and cells specifically regulate bidirectional traffic by increasing run-lengths of the organelles toward the plus- or minus-ends. Intracellular pathogens also control microtubule traffic to achieve both nuclear uptake and cell egress. Elucidation of how cytoskeletal traffic is controlled by pathogens represents important and exciting research. Other important questions to be answered include: how do competing motors coordinate their activities? How do motor protein receptors function to deliver their cargo? How are receptor and ligand physically sorted? The goal of studying endocytic sorting should be to understand the processes in a rigorous, quantitative manner such that they can be recapitulated in vitro and modeled mathematically. As an example, modeling of reaction rates combined with structural and biochemical studies has allowed for impressive understanding and in vitro recapitulation of complicated processes of actin polymerization [121]. Microscopy, especially live cell microscopy, is a powerful avenue for new experimental results. Often, the difficulty with this technique lies in interpretation and standardization of the data. It would be particularly helpful to have a

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Fig. 1. Roles of the cytoskeleton and motor proteins in endocytic sorting. The figure represents an overview of endocytic processing events that involve the cytoskeleton, as discussed in the text. A piece of cytoplasm and extracellular space is drawn along with endocytic organelles (shaded gray), microtubules (red texture with plus-ends marked), actin filaments (green) and motor proteins. The cell has undergone endocytosis followed by EE fusion, EE fission and LE fusion with lysosomes. Viral trafficking, plus-end tracking of CLIP-170, myosin V-driven endosome movement and endosomal actin rocketing are also illustrated. Relevant references are indicated in brackets. Rab protein participation in motordriven endocytic processing is also listed. Arrows and multiple motor proteins indicate the bidirectional nature of endocytic processing. Representations are not to scale.

convenient means to calibrate (at least estimate) fluorescence intensity in terms of number of fluorescent molecules. Combining live cell microscopy with optical biochemical and biophysical techniques appears promising [141].

10.1. Domain displacement overexpression Many insights have come from the use of ‘‘domain displacement’’ techniques, where a binding domain of a protein is expressed in the absence of

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the functional domain to which it is normally connected. Overexpression of this domain displaces the endogenous protein, resulting in a functional knockout. This technique has worked extremely well for the overexpression of the dynactin subunit, dynamitin [36] as well as many other proteins. Caution must be used in interpretation of overexpression experiments as phenotypes may result from both ‘‘positive’’ effects of an effector domain, or ‘‘negative’’ effects of displacing endogenous protein interactions. 10.2. Drug delivery and disease The improper transport of cellular cargo has been implicated in human diseases especially neurodegenerative diseases such as Alzheimer’s disease, amyotrophic lateral sclerosis and Charcot – Marie – Tooth disease. Motor proteins, as we have discussed, make up the primary agents for the transport of cellular cargo, and motor protein mutations can serve to model diseases of neurodegeneration [142,143]. Motor proteins and motor protein receptors are also receiving attention as targets for drug development as the motor protein and its receptor are putatively responsible for the localization and hence activity of proteins and other bioactive molecules [144]. Motor protein-based endosomal traffic can be an important consideration for drug delivery to intracellular destinations. For instance, delivery of DNA and other molecules that have been encapsulated in liposomes occurs through endocytosis, and control of microtubule-based endocytic sorting can increase drug delivery. Treatment of cells with either Taxol, which stabilizes microtubules, or nocodazole or colchicine, which depolymerize microtubules, has increased gene transfection efficiency, apparently by blocking transport of endosomes to lysosomes, and therefore preventing DNA degradation [145,146]. In related technology, one potential means to overcome multidrug resistance has been to surround the drug (e.g. digoxin) in a targeted liposome and allow the drug to be taken up by receptor-mediated endocytosis [147]. Further understanding of microtubule-based intracellular endosomal traffic could lead to the ability to specifically decrease or increase the half life of drugs within the body.

10.3. Drugs that inhibit specific motor proteins Can reagents be developed that inhibit specific events of microtubule-based motility that could, for instance, prevent the spread of an infectious agent? Mitchison and others have advocated the use of small molecule chemical inhibitors to understand cytoskeletal function [26] and have recently found that an inhibitor of myosin II inhibits cytokinesis, cell motility and cell blebbing [148], and another small molecule, monastrol, specifically inhibits the mitotic kinesin, Eg5 and arrests cells in mitosis. A similar approach to endocytic processing applied to in vitro and cell biological assays could prove rewarding. Acknowledgements This work was supported by NIH grants DK41918 and DK41296. References [1] I. Mellman, Endocytosis and molecular sorting, Annu. Rev. Cell Dev. Biol. 12 (1996) 575 – 625. [2] S. Mukherjee, R.N. Ghosh, F.R. Maxfield, Endocytosis, Physiol. Rev. 77 (1997) 759 – 803. [3] M. Marsh, H.T. McMahon, The structural era of endocytosis, Science 285 (1999) 215 – 220. [4] M. Forgac, Structure, function and regulation of the vacuolar (H+)-ATPases, FEBS Lett. 440 (1998) 258 – 263. [5] J. Harford, K. Bridges, G. Ashwell, R.D. Klausner, Intracellular dissociation of receptor-bound asialoglycoproteins in cultured hepatocytes. A pH-mediated nonlysosomal event, J. Biol. Chem. 258 (1983) 3191 – 3197. [6] J. Harford, A.W. Wolkoff, G. Ashwell, R.D. Klausner, Monensin inhibits intracellular dissociation of asialoglycoproteins from their receptor, J. Cell Biol. 96 (1983) 1824 – 1828. [7] A.W. Wolkoff, R.D. Klausner, G. Ashwell, J. Harford, Intracellular segregation of asialoglycoproteins and their receptor: a prelysosomal event subsequent to dissociation of the ligand – receptor complex, J. Cell Biol. 98 (1984) 375 – 381. [8] E. Bananis, J.W. Murray, R.J. Stockert, P. Satir, A.W. Wolkoff, Microtubule and motor-dependent endocytic vesicle sorting in vitro, J. Cell Biol. 151 (2000) 179 – 186. [9] J.A. Oka, P.H. Weigel, Microtubule-depolymerizing agents inhibit asialo-orosomucoid delivery to lysosomes but not its endocytosis or degradation in isolated rat hepatocytes, Biochim. Biophys. Acta 763 (1983) 368 – 376. [10] M. Bomsel, R. Parton, S.A. Kuznetsov, T.A. Schroer, J. Gruenberg, Microtubule- and motor-dependent fusion in vitro between apical and basolateral endocytic vesicles from MDCK cells, Cell 62 (1990) 719 – 731.

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