Molecular motors and membrane traffic in Dictyostelium

Biochimica et Biophysica Acta 1525 (2001) 234^244

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Review

Molecular motors and membrane tra¤c in Dictyostelium Shuo Ma, Petra Fey, Rex L. Chisholm * Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611, USA Received 20 November 2000

Abstract Phagocytosis and membrane traffic in general are largely dependent on the cytoskeleton and their associated molecular motors. The myosin family of motors, especially the unconventional myosins, interact with the actin cortex to facilitate the internalization of external materials during the early steps of phagocytosis. Members of the kinesin and dynein motor families, which mediate transport along microtubules (MTs), facilitate the intracellular processing of the internalized materials and the movement of membrane. Recent studies indicate that some unconventional myosins are also involved in membrane transport, and that the MT- and actin-dependent transport systems might interact with each other. Studies in Dictyostelium have led to the discovery of many motors involved in critical steps of phagocytosis and membrane transport. With the ease of genetic and biochemical approaches, the established functional analysis to test phagocytosis and vesicle transport, and the effort of the Dictyostelium cDNA and Genome Projects, Dictyostelium will continue to be a superb model system to study phagocytosis in particular and cytoskeleton and motors in general. ß 2001 Published by Elsevier Science B.V. Keywords : Membrane tra¤c ; Molecular motor; Kinesin ; Dynein; Myosin; Actin cytoskeleton; Microtubule

1. Introduction Phagocytosis and macropinocytosis, the two major forms of endocytosis in Dictyostelium, describe the internalization of external material and plasma membrane. Endocytosis, and membrane tra¤c in general, is largely dependent on the cytoskeleton and its motor proteins. Motors are mechano-enzymes that move along the cytoskeleton tracks : myosins act upon actin, and kinesins as well as dyneins act upon microtubules (MTs). The uptake and internalization of particles involves the formation of membrane ru¥es or crowns. Ru¥e extension, formation and closure of the phagocytotic cup, and short-range movement of the newly formed vesicles through the actin cortex depend on actin and myosin. Once the endosome is internalized, intracellular transport involved in the sequential steps of processing are MT-dependent and dyneinand/or kinesin-dependent. While intracellular membrane transport of cargo over long distances was long believed to depend solely on MT-based motors, recent studies dem-

* Corresponding author.; E-mail : [email protected]

onstrate that some myosins and the actin cytoskeleton also play an important role in intracellular membrane tra¤c. Dictyostelium has long been considered to be an excellent organism to study the cytoskeleton, its motors and motility in general. Its cytoskeleton organization and related processes, such as cell motility, phagocytosis and intracellular tra¤c, are similar to those in higher organisms. Dictyostelium cells are mandatory phagocytes, showing a rate of phagocytosis or macropinocytosis 2^10 times higher than other professional phagocytes such as macrophages and neutrophils [1]. Today, in the era of modern molecular biology and biochemistry, this simple eukaryote is an ideal model organism for studying the mechanisms responsible for phagocytosis because it is easy and inexpensive to grow, and because of its combination of e¤cient biochemical and genetic approaches. For example, it is straightforward in this system to create even triple mutants in a null background. As studies of conventional myosin, unconventional myosins, dynein and kinesin show, Dictyostelium has contributed signi¢cantly to our understanding of these motors. Dictyostelium is also an excellent system for the creation of stable cell lines, in which dynamic processes can be visualized using £uorescent tags, providing an excellent opportunity to study the complex dynamics of molecular motors, the cytoskeleton and membrane tra¤c.

0304-4165 / 01 / $ ^ see front matter ß 2001 Published by Elsevier Science B.V. PII: S 0 3 0 4 - 4 1 6 5 ( 0 1 ) 0 0 1 0 9 - X

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2. Actin-based motors Myosins are a conserved class of actin-based motor proteins found in virtually all eukaryotes from yeast to man. They are mechano-chemical, actin-activated MgATPases that convert the energy of ATP hydrolysis into movement. Most myosin heavy chains (HCs) consist of three domains: (1) the N-terminal globular head or motor domain, comprising the ATP binding pocket and an actin binding site; (2) the neck region, where myosin light chains (LCs) or calmodulin bind, which serves as a lever arm, and (3) the C-terminal tail, which de¢nes subclasses. Some tail domains form coiled coils, which allow the molecules to dimerize. Tails of unconventional myosins are believed to serve as cargo binding domains. Myosins have been found to play a role in many diverse cellular tasks such as muscle contraction, cell movement, cytokinesis, endo- and exocytosis, mRNA transport and vesicle transport. The large myosin superfamily is classi¢ed into at least 15 distinct groups based on phylogenetic analyses of the conserved head domain sequences. Conventional ¢lamentforming myosins belong to class II, and single or doubleheaded unconventional myosins belong to all other classes. While there are still some unclassi¢ed myosins in Dictyostelium, the characterized myosins fall into four di¡erent classes: class II or conventional myosin, and the unconventional class I, class VII and class V or XI myosins. 2.1. Dictyostelium has taught us much about conventional myosin The most studied myosins are the conventional or class II myosins. This double-headed molecule is composed of two HCs, and two pairs of essential and regulatory LCs. The LCs are thought to stabilize the neck region, where they may de¢ne the mechanical properties of the lever that ampli¢es the conformational changes produced by ATP hydrolysis. In addition, they regulate the enzymatic activity of the motor. The HC tail consists of an K-helical coiled coil able to form a parallel dimer, that in turn can self-associate into bipolar, thick ¢laments. This enables myosin II to operate in huge ¢lament arrays to drive high speed motility. While vertebrates express many myosin II isoforms, we have learned much about this motor protein from studies using Dictyostelium, which expresses only one isoform. Conventional myosin was isolated from Dictyostelium by Clarke and Spudich in 1974 [2] and 20 years later it was the second myosin to be crystallized [3,4]. This provided the basis to investigate the `swinging lever arm' model of myosin movement, which postulates that ATP hydrolysis produces a swing in the neck region, whose reversal is thought to generate force [5]. In vitro motility assays using di¡erent truncations of the neck region [6], arti¢cial lever arms [7], or LC mutations [8] indeed showed

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that alterations of the lever arm signi¢cantly a¡ected velocity. Dictyostelium myosin II localizes to the cleavage furrow of dividing cells, to the posterior of migrating cells [9,10] and to the tips of retracting pseudopods [11], consistent with a role in cell migration and cytokinesis. Dictyostelium was the ¢rst organism in which myosin II function was deleted [12] or inactivated by antisense expression [13], causing severe cytokinesis and developmental defects. Surprisingly however, the cells remained viable. Only when grown in suspension did the mutants fail to divide indicating that cytokinesis does not always require myosin II function. This interesting result has been extensively discussed in recent reviews [14^16]. Deletions of the myosin regulatory and essential LCs were also ¢rst accomplished in Dictyostelium producing defects similar to HC deletions [17,18] demonstrating the importance of all three subunits. Much research focuses on myosin regulation, the residues that can be phosphorylated and the responsible kinases (reviewed in [19,20]). Regulatory LCs have conserved phosphorylation sites (Thr18 and Ser19 in mammalian cells, Ser13 in Dictyostelium), but the physiological importance of regulatory LC phosphorylation remains questionable. While regulatory LC phosphorylation mutants in Dictyostelium [21] and yeast [22] were able to rescue the null phenotype, LC phosphorylation was found to be essential for Drosophila oogenesis [23]. The assembly state of Dictyostelium myosin is regulated by three threonines at the carboxy-terminal end of the HC [24^26]. In mutants unable to phosphorylate their HC, myosin over-assembled in the cytoskeleton [25], producing de¢cient chemotaxis [27]. When the three threonines were converted to aspartates (3UAsp-myosin) mimicking the phosphorylated state, mutants failed to rescue the myosin null phenotype, and electron microscopy showed that the 3UAsp-myosin was mainly in the dimeric bent formation, unable to assemble [28]. Recently, Nock et al. identi¢ed a single one of these phosphorylation sites as the most critical for ¢lament assembly and myosin II in vivo function [29]. 2.2. Is myosin II a vesicle transporter? Although the role of myosin in vesicle transport is strongest for other actin-based motors, there is some evidence for myosin II as a vesicle transporter. In mammalian cells, immunohistochemical evidence shows that myosin II is associated with Golgi membranes during vesicle budding [30,31]. In Dictyostelium, myosin II has been demonstrated by electron microscopy to be associated with small cytoplasmic vesicles [32]. Soll et al. found myosin II to be necessary for rapid intracellular particle movement [33,34]. However, myosin II's exact role in vesicle movement remains unknown. By shaping the actin network myosin II may be important for maintaining the

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cytoskeleton's integrity. Thus, it is possible that myosin II a¡ects particle movement only indirectly. 2.3. Unconventional myosins on the rise The unconventional or non-¢lament-forming myosins comprise at least 13 di¡erent classes. Some of the myosin classes have been found exclusively in certain organisms or cell-types, while others are ubiquitously expressed, and many cells express multiple members of several classes. This suggests that some myosins have specialized, and some have partially overlapping or common functions. These diverse myosin types are implicated in controlling cortical tension, cell migration, chemotaxis, endo- and phagocytosis and organelle transport. After the discovery and successful knock-out of myosin II in Dictyostelium, and because cells devoid of this conventional myosin still display many motile activities, researchers set out to ¢nd unconventional myosins in this organism [35^39]. This resulted in the identi¢cation of 13 unconventional myosins to date, 10 of which have been, or are in the process of being characterized. Among those are seven class I myosins (myoA^F, myoK), one class VII myosin (myoI), a class V or XI myosin (myoJ) and one at present unclassi¢ed myosin (myoM). Studies of these motors have begun to reveal interesting biological functions. 2.4. The large myosin I family The largest group of unconventional myosins is class I, consisting of single-headed, non-¢lamentous myosins. Originally discovered in Acanthamoeba in 1973 [40], in 1985 Cote et al. found the ¢rst Dictyostelium myosin I [41]. Myosin Is consist of a single HC and one to six LCs that bind to the neck region. The myosin I tails can be divided into three tail-homology domains (TH1^TH3). TH1 comprises a basic phospholipid binding domain; TH2, also called GPA or GPQ for its high bias towards Gly, Pro and either Ala or Gln, contains a secondary, ATP-independent, actin binding site; and TH3 is composed of a SH3 (Src homology 3) domain. Of the seven myosin Is expressed in Dictyostelium, three are full-length or so-called amoeboid myosins (myoB, C and D), three have a shorter tail (TH1 only; myoA, E and F), and myoK has a large head insertion but no LC binding motifs and the shortest tail of all known myosins [38,42]. In contrast to myosin II, myosin I has been localized to the leading edge of migrating Dictyostelium [43,44], suggesting a function in cell locomotion. Phenotypes of cells lacking either myoA or myoB have been shown to resemble each other. Mutants moved with reduced velocity, formed more pseudopods, and turned more frequently than wild-type [45^47]. Double mutants (A-/B-, B-/C-) showed that these myosins contribute to the generation of cortical tension [48]. These results demonstrate how

each of the numerous myosin Is is a player in the organization of the actin cytoskeleton. There is evidence that the SH3 domain (TH3) might be necessary for amoeboid myosin I targeting and/or function. In yeast it has been reported that the SH3 domain is necessary for myo5p (a class I myosin) function and contributes to its localization [49]. In Dictyostelium, the SH3 domain is not responsible for myoB location, but it is essential for myoB function in vivo [50]. 2.5. Myosin I and endocytosis Dictyostelium often has been regarded as a `mini-macrophage' whose livelihood depends on e¤cient endo- and phagocytosis. Because Dictyostelium is an e¤cient phagocyte it recently has been suggested to be a useful system to study host^pathogen interactions [51]. In this work, the intracellular pathogen Legionella pneumophila normally growing in amoeba or macrophages was shown to grow by the same mechanisms in Dictyostelium. Both macropinocytosis and phagocytosis in Dictyostelium occur by a mechanism involving actin-rich membrane ru¥es or crowns, forming the phagocytic cup. Like cell movement, phagocytosis uses the actin cytoskeleton to extend pseudopods, therefore it is not surprising that myosin motors are involved in this process. Dictyostelium myoB was the ¢rst myosin to be located to the phagocytic cup [43]. It was shown to be associated with membrane ru¥es [52]. Cells de¢cient in myoB were found to have a reduced rate of phagocytosis [53,54]. Over-expression of myoB resulted in decreased macropinocytosis, leading to the suggestion that an excess of myoB cross-links the ¢lamentous actin in the cortex too tightly, preventing the formation of large actin-rich projections [55]. Single myoC mutants were shown to have a decreased initial rate of £uid-phase pinocytosis [54]. Various double and triple mutants (A-/B-, B-/C-, B-/D-, B-/C-/ D-) had even slower doubling times than single mutants [52,54], suggesting an additive decrease in £uid-phase uptake. These results suggest, that myosin Is share in supporting endocytosis, but that their functions are not simply redundant since myoB and myoC single deletions each show a clear endocytosis defect. Findings in other organisms are in good agreement with data coming from Dictyostelium studies. Acanthamoeba myosins IB, and to a lesser extent IA, have been localized to phagocytic membranes [56]. Recently, Voigt et al. demonstrated in the pathogenic amoeba Entamoeba histolytica, that myosin IB is involved in the phagocytosis of human erythrocytes. Over-expression of myosin IB caused a severe decrease in erythrocyte phagocytosis, and myosin IB was shown to be enriched in the phagocytotic cup [57]. Dictyostelium myoK, which represents the newest and seventh member of the Dictyostelium class I myosins, seems to contribute, similar to other myosin Is, to normal cell motility and cortical tension. When Schwarz et al.

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deleted or over-expressed the myok gene however, mutants displayed more severe changes in morphology than other myosin I single knock-outs. Mutant cells showed excessive membrane ru¥ing and enlarged lamellipodia, suggesting a general disorganization of the actin cytoskeleton. In addition, these mutants had a 30% decrease in the initial rate of yeast phagocytosis, suggesting a role for myoK in the early stages of particle uptake [58]. 2.6. Myosin VII and phagocytosis Mutations in myosin VIIa in mice cause the shaker-1 phenotype, whereas human patients with myosin VIIa mutations su¡er from Usher's syndrome IB. Mutations in humans and mice both cause deafness and vestibular (balance) dysfunction. In addition (probably because of the longer life span), Usher's syndrome patients su¡er from retinitis pigmentosa, and in their teenage years gradually go blind (reviewed by [59^61]). Myosin VIIa has been localized to the actin-rich domain of the retinal pigmented epithelium (RPE) [62]). A key feature of the retina is its continuous renewal of photoreceptor cells. The RPE absorbs the shed outer segments, thereby removing otherwise toxic products, presumably through phagocytosis. Further evidence that myosin VIIa plays a role in endocytosis comes from the analysis of inner and outer hair cells from shaker-1 mice, which, in contrast to wild-type, do not internalize aminoglycosides [63]. Recently, by investigating the role of myoI in Dictyostelium, Titus provided the ¢rst clear evidence for myosin VII involvement in phagocytosis [64]. MyoI is the ¢rst myosin VII homolog found in lower eukaryotes. Cells devoid of myoI showed an 80% decrease in phagocytosis of yeast and latex beads. The mutants were also unable to grow on bacteria, while other actin-dependent processes like pinocytosis, exocytosis and cytokinesis were normal. Mutant cells formed normal phagocytic cups, suggesting a role of myoI in phagosome internalization. It will be interesting to investigate questions such as: what provides the remaining 20% capacity for phagocytosis (myoIB, myoIK)? What is the exact function of myosin VII in phagocytosis and how is it regulated ? These studies provide a brilliant example of how Dictyostelium can further our understanding of the mechanisms underlying human diseases. 2.7. Particle transporters: myosin V and VI Although no Dictyostelium myosin that plays a role in membrane tra¤cking has yet been recognized, we include these motors here because of their immensely interesting functions and interaction with the MT-based cytoskeleton (see below and several recent reviews [65^67]. In 1992, Kuznetsov et al. reported for the ¢rst time, that axonemal vesicles move on both MTs and actin ¢laments [68]. In recent years, class V myosins have been implicated

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in axonemal vesicle transport [69], mRNA transport in yeast [70,71], transport of mouse melanosomes [72,73] and ¢sh and frog melanophores [74,75]. Mehta et al. demonstrated that myosin V, unlike other characterized myosins, is a processive motor [76]. Processivity means the mechano-enzyme makes multiple steps during a single interaction with its track, allowing a single motor to e¤ciently move cargo. This ¢nding suggests that myosin V is well suited to serve as an organelle transporter. Myosin VI has also been suggested to participate in vesicle tra¤cking. In the Drosophila embryo it associates with particles in the syncytical blastoderm [77], and a partial loss of function mutant is defect in spermatogenesis [78]. In Snell's waltzer mice, mutations of myosin VI caused a degeneration of the sensory hair cells in the inner ear, resulting in deafness [79]. Myosin VI was found to be concentrated in the cochlea between the cuticular plate and the plasma membrane, a region of active membrane recycling [79,80]. The recent discovery that myosin VI moves towards the pointed ends of actin (a direction opposite to all other characterized myosins ; [81]) suggests, that this motor might transport cargo from the periphery (where the barbed ends are) towards the center of the cell. It will be of great interest to learn if Dictyostelium has a myosin VI homolog, or if this isoform represents a specialization unique to complex tissue structures. 2.8. Dictyostelium myoJ and other candidates Dictyostelium myoJ structurally resembles class V myosins, but phylogenetically it is most similar to Arabidopsis MYA1 and MYA2 [44,82], which have been suggested to form class XI [83]. While classi¢cation of myoJ is still a matter of debate, its function has been addressed by deleting the gene [82]. When mutants were tested for growth, endocytosis, phagocytosis and development, however, no obvious defects could be found. This indicates either functional redundancy (unidenti¢ed myosins?), or the phenotypical changes could not be detected with the methods employed. There are still some myosin loci in Dictyostelium that have been mapped only (myoH, G, L), but for which biochemical and physiological studies remain to be completed. A newly reported isoform myoM is the most divergent myosin yet described [38]. The continuous identi¢cation of previously unrecognized myosin raises the question of how many others will be uncovered as the Dictyostelium genome sequence is completed. It remains to be seen if any of these myosins plays a role in membrane tra¤cking. 3. MT-based membrane motors MT-based motility is known to be important for intracellular membrane transport including both the endocytic pathway and the biosynthetic/secretory pathway. Direc-

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tional transport along the MTs depends on MT-associated motor proteins, dynein and kinesin, which convert the chemical energy from ATP hydrolysis into movement along the MTs. These motors are unidirectional, moving towards either the plus-ends or the minus-ends of MTs. The majority of the kinesin superfamily members are plusend directed motors, although minus-end directed isoforms have recently been identi¢ed. Members of the dynein family are minus-end directed, although there exist uncharacterized cytoplasmic dynein HCs in vertebrates for which this remains to be con¢rmed. Kinesins and dyneins have been implicated in a wide range of functions, mainly for intracellular organelle transport during interphase, and for spindle function during mitosis and meiosis. Here, we will mainly focus on the role of these motors in membrane transport. 3.1. MTs MTs are 25 nm tubule-like structures formed by K and L tubulin heterodimers. Because of the head-to-tail alignment of the tubulin heterodimers, MTs have intrinsic polarity, with a fast growing plus-end and a slow growing minus-end. The Dictyostelium tubulin genes have been cloned with the genome containing single K-, L- and Qtubulin genes [84,85]. In vivo, MTs are nucleated at the MT organizing center (MTOC) to which their minus-ends are anchored. For most eukaryotic cells, MTs are organized into a radial array with the minus-ends focused on the MTOC located adjacent to the nucleus at the central region of the cell and the plus-ends pointing out to the cell periphery. Cells utilize MT both as sca¡old for maintaining the steady-state localization of the membrane organelles and as tracks for e¤cient transport of the materials between these compartments. In general, membrane organelles are arranged di¡erentially within the MT array, with the endoplasmic reticulum (ER) and early endosomes preferentially in the cell periphery near the MT plus-ends, and with the Golgi apparatus, late endosomes and lysosomes clustered near the MTOC. Dictyostelium MTs are organized similarly to other eukaryotic cells, however the density of MTs in the cytoplasm is signi¢cantly lower, making it easier to keep track of organelle movements along the MTs in intact cells. MTdependent bidirectional organelle transport has been well documented in Dictyostelium [86]. In recent years, various approaches have been utilized to study the molecular mechanisms behind directional transport and have yielded a few motility factors for membrane transport, including both dynein and kinesin. 3.2. Cytoplasmic dynein All characterized dynein family members move towards MT minus-ends. One major form of cytoplasmic dynein has been isolated from many organisms and is ubiqui-

tously expressed. It is a large multi-subunit protein complex (1.2 mDa) consisting of two HCs and several accessory subunits named intermediate chains (ICs), light intermediate chains (LICs) and LCs, according to their sizes. By electron microscopy, each dynein molecule has two large globular domains projecting from a common base, which appears to be composed of several smaller globular domains [87]. The HCs make up the bulk of the two globular heads while the accessory subunits likely lie at the base. The base of cytoplasmic dynein is thought to target the motor to its cargo while the globular heads are thought to be the motor domains that interact with MTs in an ATP-dependent manner [87]. In addition to the most abundant dynein, two other HC isoforms (DHC2 and DHC3) have been identi¢ed in cultured mammalian cells [88]. While DHC2 is a distant member of the cytoplasmic dynein family and seems to be speci¢cally related to Golgi functions, DHC3 is more similar to the axonemal dynein family and its function is unknown. Beyond expression patterns, relatively little is known about these other dynein isoforms. Dictyostelium is an especially good system for the investigation of dynein function since it contains a single dynein HC gene [89]. Dynein from Dictyostelium was ¢rst identi¢ed and isolated in 1990 by Koonce et al. [90]. The cDNAs for the dynein HC and IC were cloned and there seems to be a single copy of each gene in Dictyostelium [91,92]. Overexpression of a 380 kDa globular head domain of the dynein HC in Dictyostelium caused a collapse of the interphase MT network [93]. A similar MT defect was observed when dynein IC truncation mutants were overexpressed [94], suggesting that, in Dictyostelium, dynein activity is important for maintaining the radial organization of interphase MTs. Following MT dynamics using a green £uorescent protein (GFP)^K-tubulin fusion, Koonce et al. proposed that dynein serves as a cortical anchor for cytoplasmic MTs and functions in a force-generating capacity to a¡ect MT organization [95]. Recently, we have observed dynein signals at the cell cortex, further supporting this hypothesis (unpublished results). 3.2.1. Cytoplasmic dynein as a retrograde membrane motor Cytoplasmic dynein has numerous functions. In addition to its role in various aspects of spindle function during mitosis, dynein is thought to be responsible for most, if not all, of the minus-end directed MT-dependent organelle transport during interphase. A dynein activator complex, dynactin, is also required for dynein-driven vesicle motility in vitro [96,97] and for dynein-related functions in vivo [94,98^100]. Dynein is required for maintaining the steady-state distribution of certain organelles (including the Golgi complex, endosomes and lysosomes) and for transporting intermediates between these compartments in a retrograde orientation. Several lines of evidence have shown that dynein and dynactin are required for the perinuclear localization of

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the Golgi apparatus [99,101,102]. In Dictyostelium, overexpression of dynein IC domains believed to disrupt dynein^dynactin interaction resulted in the dispersion of the Golgi complex [94], indicating that Dictyostelium requires dynein for maintaining the distribution of this organelle. Using a virus protein (VSVG)^GFP fusion as a tracer, Presley et al. have elegantly demonstrated the transport of newly synthesized protein from ER to the Golgi complex along MTs, and that this process is dependent on dynein and dynactin [103]. Dynein-dependent retrograde transport is also critical for the endocytosis and phagocytosis pathways. Dynein antibody stains punctate structures that partly colocalize with endosomes and lysosomes in cultured mammalian cells [104]. Using an in vitro reconstitution assay, Aniento et al. have shown that the fusion between early and late endosomes is stimulated by polymerized MTs and cytoplasmic dynein, but not kinesin, suggesting that dynein is required for the vesicular transport from early to late endosomes [105]. Dynein has been shown to tightly associate with a population of ligand-containing endocytic vesicles in hepatocytes, therefore is proposed to help sorting the ligand away from the receptor [106]. The importance of dynein in endocytosis has been also suggested by in vivo functional studies, where disruption of dynactin by overexpressing its p50 subunit led to redistribution of endosomes and lysosomes to the cell periphery [99]. Endosome and lysosome dispersion phenotypes were also seen in blastocysts where the dynein HC is knocked-out [102], suggesting that dynein is required for the ongoing centripedal movement of endocytic organelles. Similarly, MT and dynein-dependent motility is also required for phagosome movement and maturation [107]. As a special example of endocytosis, it was recently shown that MT-dependent minus-end directed motility by cytoplasmic dynein is required for targeting the adenovirus to the nucleus for replication [108]. 3.2.2. Dictyostelium dynein studies As noted above, studies in Dictyostelium have shown that dynein plays a role in organizing the interphase MT network, thus it in£uences organelle organization and membrane tra¤c by a¡ecting the general organization of the MT tracks themselves. Dictyostelium dynein is also involved in vesicle transport directly. The punctate localization pattern of dynein suggests membrane association [90]. Pollock et al. have developed a method for preparing Dictyostelium extracts that support e¤cient bidirectional, MT-based vesicle transport in vitro [109]. When extracts from cells overexpressing a 380 kDa dynein HC mutant were used in this assay, the frequency of minus-end directed movement was signi¢cantly reduced, while plusend directed motility was una¡ected, con¢rming that dynein is indeed the motor responsible for minus-end directed membrane transport in Dictyostelium [109]. In dynein IC truncation mutants, the perinuclear Golgi

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localization was disrupted, con¢rming that Dictyostelium dynein has similar functions as in mammalian cells [94]. Recently, we have developed a system to follow dynein dynamics using an IC^GFP fusion, and for the ¢rst time, can observe dynein-associated organelle transport in living cells (Ma and Chisholm, in preparation), providing a powerful tool to study the regulation of dynein-dependent organelle transport. 3.3. Kinesin Members of the kinesin superfamily of MT motors are de¢ned by shared homology in their motor domains, which contain the ATP and MT binding sites. In addition to conventional kinesins, phylogenetic analysis suggests there are at least seven subfamilies of kinesin-related proteins (KRPs or KIFs), as well as many ungrouped kinesins. All but one KIF subfamily move toward the MT plus-ends. Among the eight major kinesin subfamilies, conventional kinesin and at least three other KIF subfamilies (Unc104/KIF1, KIF3 and KIF2) are involved exclusively in membrane transport. In addition, some members of two other KIF subfamilies (chromokinesin/KIF4 and KIFC) which previously were thought to function solely in mitotic or meiotic spindles, have now also been implicated in organelle transport. For simplicity, the kinesin family members are summarized in three groups, according to the relative position of the motor domain in the kinesin protein: the amino-terminal type (N-type), the middle type (M-type) and the carboxyl-terminal type (Ctype). Interestingly, the position of the motor domain seems to dictate the polarity of the movement: while the N- and M-type kinesins are plus-end directed, the C-type kinesins are minus-end directed. Several kinesin-like genes have been detected in Dictyostelium by polymerase chain reaction (PCR) [110] or biochemical assays [111,112]. Among them, full sequences and functional studies of two kinesin-like proteins (K7 and DdUnc104) from Dictyostelium have been reported [110,112], both of which appear to be membrane motors. Although not well characterized, partial sequences of several other kinesin-like proteins showed homology to members of several di¡erent KIF families, suggesting that Dictyostelium kinesins may also span a broad spectrum. 3.3.1. N-type and M-type kinesins: plus-end directed MT motors 3.3.1.1. Conventional kinesin, the prototype. Conventional kinesin was the ¢rst identi¢ed MT motor and remains the best-characterized kinesin family member. It is composed of two HCs with an N-terminal globular domain and an K-helical central region, along with a pair of globular LCs bound to the C-terminal region of the HC. While the N-terminal domain of the kinesin HC comprises the motor domain, its C-terminal tail and the asso-

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ciated LCs are thought to have regulatory and targeting function. Several distinct kinesin HC and LC isoforms have been identi¢ed. Conventional kinesin is a processive motor [113,114]. Extensive studies of this motor has greatly improved our understanding of the kinesin motor regarding the mechanism of kinesin motility, including enzyme kinetics, processivity, directionality, and activity regulation. These studies have been described in detail in recent reviews and therefore will not be covered here [115,116]. Several lines of evidence have shown conventional kinesin to be a broad-range membrane transporter. Originally puri¢ed from squid axoplasm [117], kinesin is associated with anterograde-moving membrane organelles in neuronal axons [118,119] and other membranous organelles [120,121]. Functional studies using antisense RNA expression, injection of function blocking antibodies, and mutational analysis demonstrate a role for kinesin in anterograde transport of membranous organelles during neurite outgrowth [122^124], Golgi to ER transport [125,126], and anterograde lysosome movement [127,128]. Using a PCR strategy, de Hostos et al. have identi¢ed six kinesin-related genes from Dictyostelium, named K2, K3, K4, K6, K7 and K8 [110]. K7 has been fully sequenced and functionally characterized. Its motor domain is most similar to conventional kinesin, but unlike conventional kinesin, K7 does not seem to have an extensive Khelical coiled-coil domain. Its non-motor domain does not show sequence similarity to any members of the kinesin superfamily, suggesting that K7 is a novel kinesin. K7 is expressed only during development, although K7-null cells show no gross development abnormalities. However, in the presence of wild-type cells, K7-null cells fail to localize to the prestalk zone, indicating a K7 function for Dictyostelium development. K7 localizes to perinuclear punctate structures, suggesting that it might be a membrane motor [110]. 3.3.1.2. Unc-104/KIF1 family, the monomeric kinesin. This family of N-terminal type kinesins is primarily monomeric. All members identi¢ed so far appear to be involved in membrane transport. A role for neuronal transport was originally suggested for members of this family as the Unc-104 mutation in Caenorhabditis elegans results in defects in anterograde synaptic vesicle transport [129]. Supporting this idea, KIF1A, the mouse Unc-104 homolog, was shown to be a neuron-speci¢c motor that is a good candidate for synaptic vesicle transport in axons [130]. Non-neuronal functions were suggested for two other ubiquitously expressed mouse KIF1 family members. KIF1B has been found to transport mitochondria along MTs [131]. The recently discovered KIF1C is localized primarily to the Golgi apparatus and its activity is required for the Golgi to ER membrane £ow [132]. Using an in vitro MT-dependent organelle transport assay, Pollock et al. puri¢ed two Dictyostelium polypep-

tides (245 and 107 kDa) that support plus-end directed organelle transport. Both appeared to be kinesin-like motors [112]. The sequence of the 245 kDa protein shares the highest homology with the C. elegans Unc-104 and, therefore, was named DdUnc-104. Unlike most other members of this family, DdUnc-104 functions as a dimeric motor. A knock-out of the DdUnc-104 gene resulted in a 60% decrease in the overall organelle transport in vivo and a 90% loss of the plus-end directed organelle transport in vitro. This suggests that DdUnc-104 is a bona ¢de membrane transporter and is the major MT motor responsible for plus-end directed organelle transport in Dictyostelium [112]. It is intriguing, however, that the DdUnc-104 null cells do not have any visible defect in morphology, growth or development, suggesting that DdUnc-104 is not essential for these processes. Other kinesin-like motors present in Dictyostelium are likely to perform redundant or complementary functions. 3.3.1.3. KIF4/chromokinesin and KIF2. Members in the chromokinesin family of N-type kinesin motors generally associate with chromosome arms and function as a mitotic motor for chromosome transport. One member of this family, KIF4, might also have a non-mitotic function. KIF4 is localized to punctate structures in the growth cones of young di¡erentiated neurons and cofractionates with microsomes, suggesting it might be a potential membrane transporter in neurons [133]. Murine KIF2 is a unique M-type kinesin that forms homodimers. KIF2 is highly expressed in developing neurons and KIF2-dependent anterograde vesicle transport appears to be important for the axonal extension in developing neurons [134]. While KIF2 is exclusively expressed in neuronal cells, an alternatively spliced isoform, KIF2L, is mainly expressed in non-neuronal cells. KIF2L is associated with lysosomes in mouse ¢broblasts and may play a role in the peripheral translocation of lysosomes [135]. Based on sequence similarity, the Dictyostelium kinesinrelated protein K6 is a candidate KIF2 homolog, although little is known about the Dictyostelium K6. 3.3.1.4. KIF3/kinesin-II, the heterotrimeric kinesin performs multiple tasks. Kinesin-II isolated from various sources consists of a heterotrimeric complex containing two di¡erent, yet related HC subunits that are members of the KIF3A/KIF3B or KRP85/95 subfamily of N-terminal kinesins. These motor subunits are structurally similar to conventional kinesin, containing a globular N-terminal motor domain linked to a C-terminal globular domain via an K-helical coiled-coil stalk. The motor subunits typically associate, via the C-terminal tail, with a non-motor subunit, the kinesin-associated protein, with a 1:1:1 stoichiometry [136,137]. Members of this family are involved in a diverse range of organelle transport in a variety of specialized cells. In Chlamydomonas and Tetrahymena, kinesin-II drives ante-

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rograde intra£agellar transport [138,139]. In Drosophila and higher animals, members of this family are mainly involved in anterograde axonal transport of membranous vesicles [140,141]. Interestingly, kinesin-II also turns out to be the long-sought-after motor for the plus-end transport of pigment granules in Xenopus melanophores. Not only does kinesin-II co-purify with the melanosome fraction [142], but expression of a dominant negative motor subunit of kinesin-II disrupts speci¢cally the dispersion of the melanosomes [143], suggesting that kinesin-II is indeed the motor responsible for melanophore dispersion. In addition, kinesin-II also has a more general function. It is ubiquitously expressed in most tissues in Xenopus, and is involved in the transport of tubular^vesicular elements between the ER and the Golgi apparatus [144]. Although no Dictyostelium kinesin-II homologs has been described, an EST clone from the Japanese cDNA project shows sequence similarity to the middle coiled-coil region of Drosophila KLP68, making it an interesting candidate for Dictyostelium kinesin-II. 3.3.1.5. Rabkinesin6, a link to Rab-regulated membrane tra¤c. The Rab family of small guanosine triphosphatases (GTPases) plays an essential role in the targeting and fusion of transport vesicles with their acceptor membrane [145]. In a search for putative e¡ectors that interact with the GTP-bound form of Rab6 (a ubiquitous Rab that associates and regulates the transport within the Golgi network), a novel N-terminal type kinesin-like protein was identi¢ed. This protein, named Rabkinesin6, has been localized to the Golgi apparatus and was shown to play a role in the dynamics of this organelle [146]. This study provides an interesting link between the kinesin family of motors and the Rab family of regulatory proteins, suggesting a mechanism for control of membrane dynamics and directional vesicular transport. 3.3.2. C-type kinesins: minus-end directed kinesin family 3.3.2.1. KIFC2 ^ minus-end directed kinesin for membrane transport? Members of this subfamily have forced a change in thinking about kinesins because they are minus-end directed motors, opposite to conventional kinesin and all other KIFs. Members of this family were believed to be solely involved in spindle function until the recent discovery of KIFC2. KIFC2 was ¢rst identi¢ed from mouse brain as a novel, neuron-speci¢c C-terminal type kinesin [147,148]. It is localized to punctate structures in neuronal cell bodies and dendrites. KIFC2 associates with multi-vesicular body-like membranous organelles, suggesting that it is a potential neuronal membrane motor [147]. Considering that all previously identi¢ed C-type kinesins were minus-end directed, KIFC2 could be the ¢rst example of a minus-end directed membrane-associated kinesin. This observation challenged the traditional idea that cytoplasmic dynein was the only

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minus-end directed MT motor involved in membrane transport. However, the polarity of KIFC2 has yet to be con¢rmed by in vitro motility assays. Interestingly, there may also be a C-type kinesin in Dictyostelium. Sequence analysis of the Dictyostelium K2 gene identi¢ed by PCR [110] showed strong homology to a family of C-terminal kinesins (KatA^KatC) from the plant Arabidopsis thaliana [149,150]. The sequence of nearly fulllength K2 cDNA has con¢rmed that its motor domains are indeed located in the C-terminal part [110]. It will be interesting to see if K2 functions as a motor for the mitotic spindle or for membrane transport. 3.3.2.2. Dictyostelium kinesin-like membrane motors: more to come. While both DdUnc-104 and K7 seem likely to be membrane motors, it is unclear in which aspects of membrane tra¤c they participate, and further functional studies are required. As mentioned above, there are several uncharacterized kinesin-like genes in Dictyostelium that might also be important for membrane tra¤c. Additional motor genes are being identi¢ed through the Dictyostelium cDNA and Genome Projects. Although further sequencing and functional information is required to further de¢ne these proteins, they are potentially interesting candidates for new kinesin members in Dictyostelium. The establishment of an in vitro organelle transport assay [109] will certainly be a valuable tool to facilitate identifying new motility factors for membrane tra¤c. Together with its combined strength of facile molecular genetics, outstanding cell biology and the ability to obtain su¤cient genetically modi¢ed material for biochemical analysis, Dictyostelium seems to be an ideal system to study the mechanism of membrane transport. 4. Cooperation between the MT and actin systems For many years, MT-based and actin-based motility were considered to be two independent systems performing their separate tasks in cell locomotion and organelle transport. Recently, there is growing evidence that both systems can work together (see above and recent reviews [151,152]). The best examples of their interaction have been provided by studies in ¢sh and frog melanophores, as well as in mammalian melanocytes, where pigment granules (melanosomes) aggregate or disperse to achieve color changes. Previously thought to be exclusively based on transport along MT, two groups showed that melanosomes can also travel along actin ¢laments [74,75]. Moreover, myosin V, in addition to kinesin-II and dynein, has been localized to melanosomes [73,75], providing a molecular basis for such organelles to travel on both cytoskeleton tracks. Melanosomes are specialized lysosomes and therefore it comes as no surprise that the same trend holds true for other organelles. For example the Golgi complex, the ER and lysosomes are associated with both MT-based

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and actin-based motors. While MTs are believed to be generally responsible for long-range transport, actin-based systems are thought to be in charge of short-range transport. Concerted action between the two systems would ensure precise control of vesicle distribution and transport. How are MT- and actin-based systems coordinated to allow a smooth transition of organelles between the two ¢lamentous tracks ? Recent discoveries suggest some interesting possibilities. In addition to forming a cross-bridge between the two cytoskeleton tracks, motors of the two systems might be functionally linked. Myosin V and conventional kinesin have been shown to directly interact [153], raising the possibility that MT- and actin-based motors might coexist in a multi-motor complex transporting cargo along both ¢lamentous systems. In addition, dynein and myosin V share the same 8 kDa LC subunit (LC8), providing a potential way for cargo to switch between di¡erent tracks [154]. In Drosophila, it was shown that myosin VI associates and colocalizes with a homolog of CLIP170, a protein that links cytoplasmic vesicles to the plus-ends of MTs [155]. Interestingly, CLIP170 also colocalizes with the dynactin complex at the MT plus-ends, providing an indirect link between myosin VI and dynein. Such direct or indirect interactions between these motors might facilitate coordination of vesicle transport along both MT and actin tracks. With our advanced understanding of myosins, and the emerging knowledge about dynein and kinesin(s), this is an exciting time to study the interactions in Dictyostelium. 5. Conclusions and future questions Both phagocytosis and intracellular tra¤c are complex processes involving multiple players. While increasing numbers of motors playing a role in these processes are being discovered, many questions concerning their function and regulation remain unclear. For example, what is the exact role of Dictyostelium myoI in phagosome internalization? Do di¡erent myosins work together in phagocytosis, and if they do, how? More generally, how is a motor targeted to the correct site of action and how is motor activity controlled? For motors with diverse functions, how do they adapt to di¡erent cargos? Using di¡erent subunit isoforms or functional adapters could provide a solution. On the other hand, many organelles are associated with motors from di¡erent families ; in this case, how do multiple motors coordinate with each other to achieve directional movement? One motor could be dominant over the others when coexisting on the same cargo. Alternatively, motor activities may be di¡erentially controlled by a common mechanism, such as phosphorylation and dephosphorylation. To facilitate coordinated action, signaling mechanisms that control membrane tra¤c, such as small GTPases, might also be good candidates for motor regulation.

Dictyostelium provides an excellent system to address these questions because of the ease with which molecular genetics and biochemical approaches can be combined. The Dictyostelium Genome and cDNA Projects have already begun to facilitate genetic analysis. The amoebae are well suited for live cell imaging, which is a valuable tool to observe motor action in vivo. For instance, because the MTs in Dictyostelium are sparse in comparison to mammalian cells, the amoebae have been proven to be superb cells for in vivo observation of MT-associated movement. Taken together, these qualities make Dictyostelium a choice organism to study phagocytosis in particular, and molecular motors and the cytoskeleton in general.

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