Organization of organelles and membrane traffic by microtubules

Organization of organelles and membrane traffic by microtubules Nelson B Cole and Jennifer Lippincott-Schwartz National Institute of Child Health and H u m a n D e v e l o p m e n t , Bethesda, USA Organelles of the central membrane system of higher eukaryotes have been shown to utilize microtubules both for maintenance of their characteristic spatial distributions and for efficient transport of their protein and lipid to diverse sites within the cell. Recent work addressing the mechanisms that underlie this organization provides new insights regarding the roles of microtubules and rnicrotubule motors in influencing organelle dynamics and specific membrane traffic routes through the cytoplasm. Current Opinion in Cell Biology 1995, 7:55-64

Introduction Eukaryotic cells have developed highly regulated membrane trafficking pathways that function to mediate exchange o f protein and lipid between distinct membranebounded compartments or organelles. Transport intermediates that utilize these pathways must often travel significant intracellular distances to reach specific target organelles. Evide'nce accumulating on the physical properties of the cytoplasm suggests that this regulated mode of transport occurs within an architectural framework that is likely to impose significant constraints on the diffusion o f macromolecular components within the cytoplasm [1]. It is not surprising, therefore, that cytoskeletal elements, particularly microtubules and their associated motor proteins, play fundamental roles not only in facilitating the selective delivery of transport intermediates between spatially segregated organelles, but also in determining the steady-state localization of the organelles themselves. In many cell types, microtubules are nucleated during interphase from a perinuclear microtubule-organizing center (MTOC) and radiate out toward the cell periphery to form an extensive network throughout the cytoplasm. Organdies of the central membrane system and their transport intermediates differentially distribute within this microtubule array, with the endoplasmic reticulum (ELL) and early endosomes preferentially distributed toward the plus (fast growing) ends of microtubules in the cell periphery, and the Golgi, late endosomes and lysosomal membranes clustered near the minus (slow growing) ends of microtubules in the vicinity of the nucleus (see Fig. 1). Such an arrangement is likely to facilitate communication between secretory and endocytic membrane transport pathways, and to promote delivery of secretory

products to specific sites. In the absence ofmicrotubules, this spatial arrangement is lost and membrane traffic between organelles and to the cell surface becomes much less efficient. Although it is generally acknowledged that microtubules and their associated proteins play fundamental roles both in the organization of organdies and in the efficient transport of protein and lipid between these structures in higher eukaryotic cells, how they accomplish this is far from being understood. Recent work focusing on this question and reviewed here has addressed an array of specific issues relating to microtubules, membrane traffic and organelle localization. These include organelle-microtubule interactions and their relationship to organdie dynamics; the roles of microtubules and microtubule motors in the membrane trafficking pathways between organelles; and mechanisms for regulating organdie motility. Because organelles do not exist as isolated entities but operate within dynamic secretory and endocytic membrane systems which are engaged in membrane traffic, these issues will be discussed in the context of the membrane pathways through which organelles communicate.

Microtubules and organelle dynamics The central membrane system of eukaryotic cells has often been described as vacuolar, with organelles such as the Elk, Golgi, endosomes and lysosomes exhibiting defined shapes and occupying stable positions within the cytoplasm. Careful examination of a variety of cell types, however, has shown that extensive and dynamic tubule structures arise from all organelles under both normal

Abbreviations BFA--brefeldin A; ER--endoplasmic reticulum; MTOC--microtubule-organizing center; TfR~ransferrin receptor; TGN--trans-Golgi network.

© Current Biology Ltd ISSN 0955-0674

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Golgi stacks

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Centrioles

and pharmacologically induced conditions (see Fig. 2) and that microtubules and microtubule motors enable these structures to extend through the cytoplasm. For example, the anastomosing tubular network characteristic of the ER is generated from extensions of ER membranes along microtubules [2]. Likewise, early endosomes [33, lysosomes [4] and the Golgi complex [S] have each been shown to have the capacity to form extended tubular processes that move along microtubules. Studies with the drug brefeldin A (BFA), which reversibly dissociates from membranes a complex of peripheral membrane proteins involved in regulating membrane traffic [h] and induces the rapid tubulation of Golgi, endosoma1 and lysosomal membranes [7,8], have revealed that membrane tubules can also serve as transport intermediates in the pathways through which organelles communicate. For example, tubules arising from the Golgi stacks and endosomes during BFA treatment have been

Fig. 1. Model for the organization of organelles and membrane transport pathways and their relation to centroromally arranged microtubules in non-polarized cells. The ER and peripheral early endosomes are distributed near the plus ends (+) of microtubules, whereas the Golgi complex (including the cis-Golgi network [CGNI, Colgi stacks and the IGN), late endosomes and lysosomes are frequently distributed near the minus ends of microtubules where centrioles are localized. Many of the membrane transport pathways connecting these organelles have been shown to be facilitated by microtubules. In the early biosynthetic system, ER-to-Go&i transport from peripheral ER sites into the central Golgi region is thought to involve microtubule minus end directed movement of the intermediate compartment (IC) while Cotgi-toER transport (observed in cells treated with BFA) involves microtubule plus end directed movement of Colgi membrane back into the ER. In the endosomal system, movement of endocytic carrier vesicles (ECV) from peripheral endosomes to late endosomes has been shown to involve a microtubule minus end directed mechanism.

shown to enhance membrane traffic from the Golgi to the ER and between endosomes and the Iruns-Golgi network (TGN) respectively, without causing non-specific fusion among organelles [6]. Although the extent of organellar tubulation in unperturbed cells is unknown, it is important to emphasize a possible major role for tubular processes in facilitating membrane exchange throughout the cytoplasm. The dynamic morphological characteristics of organelles are coupled to their capacity to move bidirectionally on microtubules (both plus and minus end directed movement). Golgi membranes are normally localized at the minus ends of microtubules in the perinuclear region of non-polarized cells, In the presence of BFA, however, extensive Golgi tubules form that extend along microtubules toward their plus ends. Similarly, BFA treatment causes membranes of the TGN first to extend toward the

Organization of organelles and membrane traffic by microtubules Cole and Lippincott-Schwartz

Fig. 2. Tubular morphology of endosomes and ER membranes. Endosomes (a) were labeled with endocytosed horseradish peroxidase and treated with BFA. ER membranes (b) were labeled with the fluorescent membrane marker DiOC 6. plus ends of microtubules (perhaps to facilitate membrane fusion with the endosomal system), and then to collapse toward the minus ends o f microtubules at the M T O C [7,8]. Other manipulations, such as cytosolic acidification in various cell types, result in the redistribution o f late endosomes [9] and lysosomes [10] toward the plus ends of microtubules, whereas alkalinization causes a shift in distribution toward their minus ends. These redistributions are often accompanied by shape changes, such as fragmentation or tubulation of the particular organelles [ 10]. Pigment granules in melanophores likewise exhibit bidirectional microtubule-dependent dispersion and aggregation behavior that appears to be regulated by phosphorylation (see below) [11,12]. These results suggest that most organelles have the capacity to bind both plus and minus end directed motors, and recent in vitro and organelle-motor competition binding studies support the concept that a regulated motor complex which contains both plus and minus end directed motor activities and shared activator molecules may control the direction of organelle movement [13,14]. As discussed below, both the microtubule plus end directed motor kinesin and the minus end directed motor cytoplasmic dynein have been shown to associate with organelle membranes and their transport intermediates, and they

are likely to play important roles in facilitating membrane exchange between specific organelles.

Microtubules and the early secretory membrane system The early secretory pathway comprises the ER, which serves as the site of synthesis of lumenal and transmembrane proteins; the Golgi complex, where secretory products are processed and sorted; and transport intermediates, which move between the E R and Golgi complex. Microtubules are known to play important roles within this system, both in organelle positioning and in traffic of vesicular and tubular intermediates.

The endoplasmic reticulum The E k comprises an extensive array of interconnecting tubules and cisternae that extend peripherally along microtubules throughout the cytoplasm. Evidence has accumulated that the extension of the E R toward the periphery of the cell and its ability to form extended

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Cytoskeleton reticular networks is attributable to a plus end directed microtubular m o t o r driven process. In vitro motility reconstitution assays using membranes from a variety o f sources [15-17] were able to generate cell-cycle and microtubule dependent reticular membrane networks similar to those observed in living cells that had been stained with the lipophilic dye DiOC6, which labels ElK membranes [2]. These and other studies have shown that ElK membranes have the capacity to form extended tubular networks in vitro, the characteristics of which are dependent on microtubule m o t o r proteins [15-17]. A recent study using rat liver E R and Golgi fractions purified from sucrose gradients showed that the majority of moving tubules contained distinct globular domains at their leading tips [18°]. Negative stain electron microscopy showed that these motile globular subdomains were enriched (primarily in the Golgi fractions) in both secretory products and microtubule-binding ability, supporting the concept that sorting o f both motor molecules and membrane contents into specialized organellar domains may be an important regulatory component of m e m brane traffic activity. These experiments, as well as those that preceded them [17], were performed in the presence of interphase Xenopus egg extracts, which presumably provide the motor and accessory factors necessary for tubule extension and network formation. It remains unclear whether components for these active processes are derived solely from the cell extracts, or also include factors attached to membranes. Evidence consistent with a role for kinesin in the plus end directed extension of the ElK has come from a variety of studies. Immunofluorescence results with antikinesin antibodies generally show punctate staining patterns which are often closely associated with ElK m e m branes [19-21]. Antibodies to kmectin, an abundant integral membrane kinesin-binding protein [22] give a reticular staining pattern characteristic of ElK staining in several cell types [22]. Finally, suppression of kinesin heavy chain expression in cultured rat hippocampal neurons by antisense oligonucleotides resulted in the retraction of the ElK network from the periphery into the center of the cell, without affecting the distribution of microtubules [23*']. In many cases, immunolocalization studies using antikinesin antibodies label punctate membrane-bound structures scattered throughout the cytoplasm that do not appear to be associated with ElK membranes (for a review, see [21]). lKecent work in normal rat kidney cells has identified these structures as pre-Golgi m e m branes which function as intermediates in the transport of protein and lipid between the ElK and Golgi complex [24"]. Transport o f these structures from peripheral sites in the cytoplasm into the central Golgi region has been shown to require microtubules [25] and would evidendy be a minus end directed process. Consistent with this, Mizuno and Singer [26*] showed that a population of stable microtubules appears to be associated with transitional elements of the ElK as well as the Golgi complex, and to be aligned with transport intermediates en route

to the Golgi complex. The association o f kinesin with pre-Golgi transport intermediates (which move in the direction of the minus end along microtubules into the Golgi region) raises the question of why a plus end directed motor like kinesin would be localized on these structures. One possibility is that kinesin travels with these membranes in an inactive state in order to localize to the Golgi complex where it could become activated for microtubule plus end directed recycling of membrane back to the ElK. Two of our observations [24"] support a role for kinesin in membrane recycling. First, perturbations in membrane traffic between the ElK and Golgi complex (including temperature manipulations and BFA treatment) suggest that kinesin constitutively cycles between the ElK and the Golgi complex. Second, microinjection of anti-kinesin antibodies had no effect on ER-to-Golgi traffic, but reduced recycling of Golgi membrane back to the ElK observed in the presence of BFA. H o w kinesin motor activity might be selectively turned on and off as it travels with membranes between the ElK and Golgi complex has not been addressed, but it could share similar features to that suggested by Hirokawa and colleagues [27,28] for cytoplasmic dynein, which has been proposed to be selectively turned on and off during fast axonal transport.

The Golgi complex The Golgi complex receives, processes and sorts lipid and protein arriving from the ElK, and consists of stacked membrane cisternae and associated tubules that utilize microtubules to actively cluster toward the cell center [29,30]. Substances that disrupt or alter microtubule structure (such as nocodazole or taxol) result in the redistribution of Golgi structures to peripheral sites throughout the cell [29,31]; these remain functional but are Ilo longer able to direct material to specific domams on the plasma membrane [32-34]. U p o n removal of nocodazole, dispersed Golgi elements recluster toward the M T O C along re-polymerized microtubules [35]. In a process reminiscent of this reclustering, cytoplasmic dynein has been implicated in the 'capture' and minus end directed movement of exogenously added Golgi membranes to semi-intact Chinese hamster ovary cells [36], and may be the motor responsible for the analogous process of clustering of transport intermediates during normal transport between EIK and Golgi. hnmunofluorescence studies using anti-dynein antibodies, however, have yet to reveal an enrichment of cytoplasmic dynein on either pre-Golgi or Golgi membranes in fixed cells. bl vitro binding assays examining the requirement for Golgi membrane binding to microtubules have shown that inactivation of cytoplasmic dynein by UV/vanadate treatment had no effect on binding, but that heat- and N-ethylmaleimide-sensitive cytosolic factors and Golgi membrane proteins were reqmred [37,38]. These restilts suggest that organelle binding to microtubules and subsequent motility along microtubules are mediated by distinct molecular components.

Organization of organellesand membranetraffic by microtubulesCole and Lippincott-Schwartz An ass.ociation of kinesin with Golgi membranes has been shown by several recent studies in rat hepatocytes [39*] and normal rat kidney cens [24*]. Two roles for kinesin on these membranes can be envisioned: the recycling of membrane back into the EP,, as mentioned above, and/or the transport o f secretory material from the T G N to the plasma membrane. Both types of transport are exaggerated by BFA treatment, which causes the formation o f long tubule processes which extend out of both the Golgi stacks and the T G N towards the plus ends ofmicrotubules. Suppression ofkinesin heavy chain expression by antisense ohgonucleotides was shown to inhibit the extension o f TGN-derived tubules in the presence of BFA [23"*]. Likewise, as described above, the extension o f BFA-induced tubules from the Golgi stacks could be inhibited by injecting anti-kinesin antibodies [24°]. Thus kinesin appears to play a role in microtubule plus end directed movements leading out of the central Golgi complex.

Role of microtubules in late secretory processes Compelling evidence for roles of microtubules and motor proteins in post-Golgi organelle transport came with the development of cell-free motility assays, initially utilizing membranes and cytosol from squid axoplasm [21,40]. More recently, various heterologous systems have demonstrated the almost universal ability of lysates containing motor proteins to support microtubule-based organelle motility. Burkhardt et al. [41"] have recently reconstituted the microtubule-dependent transport of lytic granules from cytotoxic T cells using cytosol from chick embryo fibroblasts. Isolated granules were found to bind to and translocate along microtubules at an average rate of 1 ~rn s-1. Granule motility required exogenous cytosol, hydrolyzable nucleotides and intact granular membranes, and appeared to be driven exclusively by kinesin. As the granules themselves did not bear sufficient levels of kinesin to mediate motility, these authors suggested that kinesin becomes bound to the granule membrane only after the cell is stimulated to secrete. Schmitz et al. [42"] found similar results regarding kinesin's association with isolated chromaflqn granules, which also target and release their contents via a regulated pathway and exhibit kinesin-dependent motihty in vitro, but which contain little bound kinesin. In polarized epithelial cells, microtubules are oriented with their minus ends near the apical cytoplasm and their plus ends extended toward the basal cytoplasm and Golgi complex. A dense mat of randomly aligned actin filaments also exists beneath the apical membrane (for review, see [43]). Microtubules are generally required for efficient transport of membranes to the apical surface via direct and transcytotic pathways [44], but are not required for protein transport to the basolateral surface

[34,45,46]. Apically directed secretory and membrane proteins are often missorted to the basolateral surface in the presence of nocodazole [46]. It may be expected, therefore, that a dynein-hke motor would be responsible for apically directed membrane traffic. Recently, Golgi membranes were isolated from intestinal epithehal cells and tested for possession of both microtubule- and actinbased motor molecules [47°°]. Cytoplasmic dynein and myosin-I, but not kinesin, were found on the isolated Golgi membranes. Interestingly, when the membranes were further subfractionated, immunoblot analysis indicated that dynein was found on small vesicular membranes contaiifing markers of the T G N (for example, TGN38/41), but not on Golgi stacks. Myosin-I could be found on both types of membranes, and co-pelleted with dynein in membrane-microtubule binding assays. In addition, small vesicles that contained both motors were able to bind and apparently cross-link microtubules and actin filaments. These data support the hypothesis that cytoplasmic dynein is responsible for generating microtubule minus end directed movement ofpost-Golgi vesicles to the apical cortex and that myosin-I provides the subsequent force for vesicle dehvery to the apical membrane through an actin-rich meshwork [47"°].

Microtubules and the organization and traffic of the endosomal system In the endocytic pathway, soluble and receptor-bound molecules are internalized from the plasma membrane and delivered to acidified early endosomal compartments, where receptor and ligand dissociate. Ligands and solutes are generally sorted to late endosomes and lysosomes to be degraded, whereas receptors may recycle back to the plasma membrane directly or via the T G N [48]. It is clear that endocytosed material is translocated from peripheral to perinuclear sites as it progresses through endocytic organelles en route to lysosomes [49,50], and that this process is dependent on microtubules [51,52]. What is not clear, however, are the exact pathways followed by soluble and receptor-bound molecules moving within these organelles and the role o f microtubules and motor molecules in facilitating this traffic. Nevertheless,. studies using diverse approaches, including video microscopy [4,53] and in vitro fusion assays [48], have supported the view that the endosomal system comprises distinct early and late compartments, each of which is a highly dynamic, intercommunicating membrane system continually engaging in 'homotypic' fusion events. Early endosomes are thought to be formed continually via the coalescence of vesicles derived from the plasma membrane [54]. The generation of domains within these membranes for sorting proteins is likely to require the activity of vacuolar ATPases [55], and it has been suggested that this is influenced by microtubule motor dependent processes [56]. Microtubules do not appear to be

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60 Cytoskeleton required for receptor recycling back to the cell surface, however [57°], nor does microtubule disruption affect homotypic fusion o f early endosomes with each other [48,58°°]. The abihty o f molecules to be delivered from early to late endosomal membranes has been shown by Gruenberg and colleagues [51] to depend on microtubules; it is mediated by 2 0 0 - 4 0 0 n m endosomal carrier vesicles which do not undergo self-fusion or fusion with early endosomes in vitro [51]. These vesicles are capable o f fusing with late endosomes, however, in a process which is stimulated by microtubules, microtubule-associated proteins, and cytoplasmic dynein, but not by kinesin [58°°]. Molecular components that mediate carrier vesicle association with microtubules have begun to be characterized in vitro [59,60]. These studies found that such associations were trypsin-sensitive, independent of cytoplasmic dynein and kinesin, and were dependent on the presence of cytosol and CLIP-170, a cytoplasmic phosphoprotein that has been proposed to link carrier vesicle membranes to microtubules [59,61]. The function o f CLIP-170 may not be restricted solely to the binding of endosomal carrier vesicles to microtubules, but may be involved in the microtubule-dependent organization of the early endosomal system, as antibodies to CLIP170 co-localize with a subset of endocytic structures displaying the transferrin receptor (TfR) [59], generally considered as a marker for early endosomes. In addition to playing a crucial role in membrane traffic between early and late endosomes, microtubules are involved in the spatial organization of these two membrane systems. The early endosomal system, which extends from the cell periphery into the perinuclear region, also undergoes microtubule-dependent spatial rearrangements upon BFA treatment [7,8]. Short exposures to BFA result in extensive fusion and tubulation of early endosomes towards the periphery o f ceils, whereas during longer exposures (30 min or more)- early endosomes undergo a reversible microtubule-dependent retraction into the centrosomal region (see Fig. 3). The late endosomal

system normally comprises discrete structures (including late endosomes and lysosomes) that are clustered near the perinuclear region. These structures have been found to undergo reversible microtubule-dependent extension and retraction upon changes in cytoplasmic pH, as well as upon treatment with BFA [7,9,10]. Suppression of kinesin heavy chain expression inhibited the plus end directed movement of endosomal membranes induced both by low pH and by treatment with BFA [23"*], consistent with earlier reports which described a requirement for kinesin in the plus end directed extension of lysosomal tubules in macrophages [62]. An intriguing effect of microtubule disruption on the uptake of TfR from the cell surface has been observed in two studies [57°,63°]. Both found that the rate o f TfR internalization was significantly decreased in cells treated with nocodazole or colchicine compared with controls. Using photobleaching recovery techniques, Thatte et al. [63"] showed that as the endocytic rate decreased, the receptors became immobilized on the cell surface. These results suggest that lateral movement of Tflks (which may underlie their ability to enter endocytic structures) is affected by the status o f microtubules. Whether this effect is direct or indirect (for example, via actin or intermediate filament networks) needs to be explored further.

Mechanisms for regulating organelle movement along microtubules Although our knowledge about the structural, enzymatic and biochemical properties o f kinesin and cytoplasmic dynein has advanced rapidly [21], we still know little about how these motors and their accessory molecules associate with organelles and become activated to drive membrane movement in living ceils. For example, a number of reports on a variety o f ceil types have shown that substantial amounts of kinesin and cytoplasmic dynein exist in soluble pools [19,21,40].

Fig. 3. Early effects of BFA on the distribution of endosomes containing fluorescently labeled transferrin in normal rat kidney cells. (a) Untreated cells; (b) BFAtreated cells. Note the excessive tubulation and extension of endosomes in treated cells.

Organization of organelles and membrane traffic by microtubules Cole and kippincott-Schwartz Thus it has been proposed that the ATPase activities o f these motors must be regulated such that only productive interactions with organelles and microtubules result in their activation [64]. Staining with anti-kinesin antibodies, however, shows an almost exclusive association with membrane-bounded structures [21], Indeed, a few observations have contradicted the idea that kinesin and accessory factors are recruited from soluble pools for organdie transport [65]. It remains unclear, therefore, whether cells regulate the recruitment of soluble motors to organelle membranes, or whether these motors are stably bound and only become dissociated from organelle membranes during isolation procedures.

Phosphorylation One increasingly recognized mechanism for regulating organelle motility involves protein phosphorylation. For example, substantial evidence exists ofphosphorylationmediated changes in microtubule-dependent pigment granule motility in fish chromatophores in vivo [10,11]. In addition, agents that elevate cAMP levels were shown to increase the frequency o f linear movement of secretory vesicles in Aplysia bag cell neurons [66]; the change in frequency was recendy suggested by Azhderian et al. [67"] to be due to modulation of the linkage between organelles, motors, and microtubules. Finally, in intact CV-1 cells, drug treatments that target intracellular kinases and phosphatases resulted in major changes in microtubule-dependent vesicle motility [68"]. Addition of the phosphatase inhibitor okadaic acid, agents that increase cAMP synthesis, or fetal calf serum restilted in increase in the frequency of vesicle and particle movements, as well as velocity and run lengths, without apparent effects on microtubules or on the overall distribution of outward versus inward movement [68"].

kinesin-associated proteins to changes in motor activity [74"], Cytosolic extracts treated with okadaic acid, previously shown to increase vesicle and particle motility in intact cells ([68"], see above), generated increased kinesin motor activity in both granule motility and microtubule ghding assays in a phosphorylation-dependent manner, suggesting that it is kinesin binding to microtubules that is stimulated by phosphorylation. Several proteins that copurify with kinesin were hyperphosphorylated as a result of okadaic acid treatment. These include a 150 kDa protein that may be a member of the activator II complex, a likely shared activator of both kinesin and cytoplasmic dynein based organdie movement [14[, and 73 and 79 kDa proteins that had previously been found to co-precipitate with kinesin from cultured sympathetic neurons [64]. It seems reasonable to expect, therefore, that phosphorylation will be shown to modulate organelle motility by different, albeit coordinately regulated, mechanisms that are hkely to influence both motor--organelle and motor-microtubule interactions.

Cytosolic coat proteins As mentioned earlier, treatment of cells with BFA induces the rapid formation and microtubule-dependent extension of tubular structures which emanate from several organdies within the central membrane system, including the Golgi complex, TGN/endosomes, and lysosomes. The molecular basis for the effects of BFA on the Golgi complex, for example, has been shown to he in the abihty of BFA to inhibit the binding to Golgi membranes of specific cytosolic coat protein complexes (coatomer) which normally act to maintain Golgi steady-state structure [6]. Binding of coatomer to membranes has been shown to be required for Eg.-toGolgi transport [75,76], which is a microtubule-facilitared minus end directed process. By contrast, Golgi-toElk traffic occurs in the absence of coat binding (during BFA treatment) by a microtubule plus end directed process. Coatomer binding therefore could directly or indirectly affect the activities of motor proteins associated with these membranes. Additional BFA-sensirive compartment-specific coat complexes are likely to be identified on TGN/endosomes and lysosomal membranes, and these could also modulate the microtubule motor activities associated with these membranes.

Although suggestive, these studies have not yet correlated in vivo changes in organdie motihty with changes in the phosphorylation status of microtubule motors or their associated proteins. Both the heavy and light chains o f kinesin have been shown to be phosphorylated in vivo [21,64,69], as has the putative kinesin receptor, kinectin [64]. Likewise, current evidence suggests that the heavy, intermediate, and light chains o f cytoplasmic dynein are phosphorylated in vivo [70"',71"]. Interestingly, treatment of cultured fibroblasts with okadaic acid mimicked the effect obtained by serum starvation [72], the apparent redistribution of cytoplasmic dynein from lysosomes to the cytoplasm, and resulted in increased phosphorylation of dynein heavy chain [70"].

Concluding remarks

Several recent reports have attempted to correlate kinesin and kinesin-associated protein phosphorylation with motor activity in vitro [69,73]. It remains unclear, however, whether in vitro kinesin phosphorylation is physiologically relevant, as its phosphorylation status remained unchanged in sympathetic neurons treated with agents that target various intracellular kinases [64]. A recent study has linked the phosphorylation state of several

Microtubules play a crucial role in the organization and dynamics of membrane organelles within higher eukaryotic cells and are required for efficient communication between these structures, enabhng cells to respond efficiently to changing membrane and secretory needs during cell growth, development and differentiation. Understanding how microtubules and their associated motor proteins control the 'geography' oforganelles and

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membrane transport pathways is a major challenge that has only begun to be addressed. Recent work has provided insight into the association o f microtubule motors with different organelles and organelle transport intermediates and the role o f microtubules in membrane transport pathways. H o w these motors and their accessory molecules specifically interact with organelles and become activated to drive membrane movement within cells, however, remains ripe for future investigation.

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NB Cole and J Lippincott-Schwartz, Cell Biology and Metabolism Branch, National Institute of Child Health and Humau Development, Natiorkal Institutes of Health, Bethesda, MD 20892, USA.