Experimental Cell Research 246, 263–279 (1999) Article ID excr.1998.4326, available online at http://www.idealibrary.com on

REVIEW Role of Microtubules in the Organization of the Golgi Complex Johan Thyberg* ,1 and Stanislaw Moskalewski† *Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, Box 285, S-171 77 Stockholm, Sweden; and †Department of Histology and Embryology, Institute of Biostructure, Medical School, PL-02-004 Warsaw, Poland

The Golgi complex of mammalian cells is composed of cisternal stacks that function in processing and sorting of membrane and luminal proteins during transport from the site of synthesis in the endoplasmic reticulum to lysosomes, secretory vacuoles, and the cell surface. Even though exceptions are found, the Golgi stacks are usually arranged as an interconnected network in the region around the centrosome, the major organizing center for cytoplasmic microtubules. A close relation thus exists between Golgi elements and microtubules (especially the stable subpopulation enriched in detyrosinated and acetylated tubulin). After drug-induced disruption of microtubules, the Golgi stacks are disconnected from each other, partly broken up, dispersed in the cytoplasm, and redistributed to endoplasmic reticulum exit sites. Despite this, intracellular protein traffic is only moderately disturbed. Following removal of the drugs, scattered Golgi elements move along reassembling microtubules back to the centrosomal region and reunite into a continuous system. The microtubule-dependent motor proteins cytoplasmic dynein and kinesin bind to Golgi membranes and have been implicated in vesicular transport to and from the Golgi complex. Microinjection of dynein heavy chain antibodies causes dispersal of the Golgi complex, and the Golgi complex of cells lacking cytoplasmic dynein is likewise spread throughout the cytoplasm. In a similar manner, kinesin antibodies have been found to inhibit Golgi-toendoplasmic reticulum transport in brefeldin A-treated cells and scattering of Golgi elements along remaining microtubules in cells exposed to a low concentration of nocodazole. The molecular mechanisms in the interaction between microtubules and membranes are, however, incompletely understood. During mitosis, the Golgi complex is extensively reorganized in order to ensure an equal partitioning of this single-copy organelle between the daughter cells. Mitosis-promoting factor, a complex of cdc2 kinase and cyclin B, is a key regulator of this and other events in the induction of cell division. Cytoplasmic microtu1 To whom reprint requests should be addressed. Fax: 468-301833. E-mail: [email protected].

bules depolymerize in prophase and as a result thereof, the Golgi stacks become smaller, disengage from each other, and take up a perinuclear distribution. The mitotic spindle is thereafter put together, aligns the chromosomes in the metaphase plate, and eventually pulls the sister chromatids apart in anaphase. In parallel, the Golgi stacks are broken down into clusters of vesicles and tubules and movement of protein along the exocytic and endocytic pathways is inhibited. Using a cell-free system, it has been established that the fragmentation of the Golgi stacks is due to a continued budding of transport vesicles and a concomitant inhibition of the fusion of the vesicles with their target membranes. In telophase and after cytokinesis, a Golgi complex made up of interconnected cisternal stacks is recreated in each daughter cell and intracellular protein traffic is resumed. This restoration of a normal interphase morphology and function is dependent on reassembly of a radiating array of cytoplasmic microtubules along which vesicles can be carried and on reactivation of the machinery for membrane fusion. © 1999 Academic Press

INTRODUCTION

Microtubules constitute a major component in the cytoskeleton of mammalian cells and play a vital role in the control of cell morphology and function. For instance, they provide physical strength for determination of the shape and internal arrangement of cells, delineate tracks along which membrane organelles are transported during exocytosis and endocytosis, and form the spindle which separates the chromosomes during mitosis [1]. Fourteen years ago, we reviewed the knowledge present at that time regarding the role of microtubules in the organization of the Golgi complex [2]. Largely based on studies of the effects of antimicrotubular drugs on cell morphology and secretion, it was concluded that intact microtubules are required to preserve the normal structure and function of the Golgi complex. After depolymerization of microtubules, the cisternal stacks are separated from each other, dis-

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persed in the cytoplasm, and structurally modified. In parallel, intracellular transport, processing, and extracellular release of secretory proteins are disturbed to a variable extent. In the succeeding years, considerable progress has been made in the understanding of both the microtubule system and the secretory apparatus. Thus, a great deal has been learned about the dynamics of microtubules [3–5] and the role of motor proteins such as dynein and kinesin in microtubule-dependent transport [6 –9]. There has also been a rapid advance in the knowledge of the machinery for intracellular protein sorting and transport and its significance for the maintenance of the organelles along the secretory pathway [10 –12]. Here, we try to summarize these developments with emphasis on the interactions of microtubules with the Golgi complex and other related membrane organelles. At the time of completion of this review, the 100th anniversary of the description of the “apparato reticolare interno” by Camillo Golgi was celebrated in Pavia, Italy. Many scientific journals have also paid attention to the birthday of the Golgi complex. For additional and more detailed information about the morphology, biochemistry, and function of this multifaceted organelle, the readers may for example be referred to the special Golgi issues published in Trends in Cell Biology (Vol. 8, 1998), Histochemistry and Cell Biology (Vol. 109, 1998), and Biochimica et Biophysica Acta (Vol. 1404, 1998). THE CYTOPLASMIC MICROTUBULE SYSTEM

Composition of Microtubules Microtubules are cylinder-shaped polymers composed of protein subunits called tubulin (heterodimers of a- and b-tubulin) and smaller amounts of several other proteins [13]. The latter are collectively referred to as microtubule-associated proteins (MAPs) and include at least three groups: (1) structural proteins which bind to and control the assembly of microtubules, (2) motor proteins which use energy generated by ATP hydrolysis to drive vesicular transport along microtubules, and (3) various other proteins with a less well defined microtubule association. Polymerization of tubulin gives rise to tubules with an outer diameter of about 25 nm and a 5-nm-thick wall in which 13 protofilaments normally can be resolved. The tubulin subunits are all oriented in the same direction and this constitutes the basis for the polarity of microtubules with a plus (fast-growing) end and a minus (slow-growing) end. Hence, the polarity of the dimers appears to be such that a-tubulin points to the minus end and b-tubulin to the plus end [14]. The synthesis of tubulin in the cell is controlled by an autoregulatory mechanism in which free tubulin subunits destabilize b-tubulin mRNA still bound to polyribosomes [15]. In ad-

dition, it has been suggested that excess of a-tubulin inhibits the translation of its own mRNA in order to ensure stoichiometric synthesis of a- and b-tubulin [16]. Organization of Microtubules by the Centrosome The number and distribution of microtubules in the cell is to a large extent regulated by the centrosome [17, 18]. This organelle usually exists in one copy per cell and is made up of a pair of centrioles surrounded by satellites of fibrogranular material. When new microtubules form, the minus end is attached to the centrosome and the plus end grows toward the periphery, so giving rise to a characteristic radiating pattern. Some years ago, g-tubulin was identified as a new and highly conserved member of the tubulin family and found to be associated with the centrosome [19, 20]. It is believed that this protein takes part in the nucleation of microtubules and the establishment of microtubule polarity [21, 22]. However, a large number of other molecular components are also found in the centrosome and their exact functions remain to be defined [23]. In each cell cycle, the centrosome is duplicated at about the same time as the DNA in the nucleus. This is a prerequisite for the assembly of a bipolar spindle and an accurate segregation of the chromosomes during mitosis. Moreover, it ensures that each daughter cell will receive its own microtubule-organizing center [18]. Dynamics and Posttranslational Modifications of Microtubules Microtubules vary in stability with a large, rapidly turning over population and a small, slowly turning over population. Furthermore, growing and shrinking microtubules occur simultaneously, a phenomenon referred to as dynamic instability [24 –26]. Hydrolysis of GTP reversibly bound to tubulin has been implicated in the shift between these phases [27, 28]. A small cap of GTP tubulin protects the growing end of microtubules and loss of this cap induces a rapid disassembly of the polymer. However, studies of living cells have indicated that local cytoplasmic factors may strongly influence microtubule dynamics and give rise to microtubules that neither grow nor shrink [29]. In addition, there exist populations of microtubules in which the tubulin subunits have been posttranslationally modified. For example, detyrosinated a-tubulin is concentrated in a limited subset of interphase microtubules and a similar situation exists with regard to acetylated a-tubulin [30 –32]. Microtubules enriched in these isoforms of tubulin have a longer half-life and are more resistant to drug-induced depolymerization than the majority of cytoplasmic microtubules and make up the slowly turning over population of microtubules mentioned above. Detyrosination or acetylation of tubulin

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is, however, not enough to make microtubules stable and it is believed that these modifications are accompanied by or occur as a result of other modifications [33, 34]. Antimicrotubular Drugs Drugs that interfere with the polymerization of tubulin have played a fundamental role in microtubule research [35]. In fact, tubulin was originally identified as the protein subunit of microtubules on the basis of its ability to bind strongly and almost irreversibly to the plant alkaloid colchicine. If cells are treated with colchicine, assembly of new microtubules is inhibited and preexisting microtubules break down into free tubulin subunits. A similar effect is obtained with the synthetic compound nocodazole [36]. However, the binding of nocodazole to tubulin is reversible [37] and a rapid recovery follows upon its removal [38]. Vinca alkaloids like vinblastine and vincristine likewise destroy preexisting microtubules, but in the presence of these drugs tubulin tends to aggregate into paracrystals instead of adding to the free pool of subunits. In contrast to the aforementioned substances, paclitaxel (Taxol) is a plant derivative that stabilizes and induces bundling of microtubules [39]. Nonetheless, the biological effects of paclitaxel resemble those of the microtubule-disruptive agents, indicating that the dynamic behavior of microtubules is essential for their functions in the cell. In clinical medicine, antimicrotubular drugs have found use because of their ability to inhibit mitosis, and particular interest has recently been paid to the application of paclitaxel in cancer treatment [40]. THE GOLGI COMPLEX

Structural Organization The Golgi complex of mammalian cells is built up of stacks of flattened cisterna, tubules, and small vesicles and takes part in modification, sorting, and transport of secretory products, lysosomal enzymes, and membrane components [41]. Utilizing a combination of cytochemical and electron microscopic techniques, it has been possible to divide the stacks into at least three subcompartments—cis, medial, and trans (Fig. 1)— that differ in composition and function [42– 45]. Threedimensional electron microscopy has further demonstrated that the stacks are linked together in a continuous system by anastomosing branches and that there exists a network of tubules and vesicles on each side of the stacks, usually referred to as the cis-Golgi network and the trans-Golgi network, respectively [46 – 48]. Using a freeze-drying and platinum replica technique, isolated Golgi cisternae were observed to consist of two discrete domains, a smooth-surfaced central region and a more roughly textured peripheral

FIG. 1. A schematic view of the secretory system of eucaryotic cells. N, nucleus; ER, endoplasmic reticulum; IC, intermediate compartment; CGN, cis-Golgi network; TGN, trans-Golgi network; MT, microtubules; PM, plasma membrane. Microtubules function as tracks for the bidirectional transport of vesicular and tubular structures between the ER and the Golgi stacks on one hand and between the Golgi stacks and the plasma membrane on the other hand.

region. The latter area displayed two types of protein coats, closely packed 10-nm particles appearing on the surface of budding vesicles and a finer coat evenly distributed over the periphery of the cisternae [49]. A dispersed tubulovesicular system, originally identified as elements in which membrane proteins pile up at 15°C [50], has also been described and suggested to serve as an intermediate linking the endoplasmic reticulum (ER) and the Golgi complex [51–53]. These structures (often referred to as ERGIC, ER–Golgi intermediate compartment, or VTC, vesicular tubular clusters) are believed to function in the transfer of proteins between the organelles in the early parts of the secretory pathway as well as the retrieval of resident ER proteins [54 –57]. Accordingly, recent studies on living cells using chimeras of viral glycoproteins and green fluorescent protein have disclosed the existence of a heterogeneous set of structures involved in ER-toGolgi transport [58, 59]. Despite the rapid advance in the exploration of intracellular protein transport during the past few years (see below), there are still large gaps in our knowledge of how the morphology of the Golgi complex is established and maintained. Early models suggested that Golgi cisternae are formed by fusion of ER-derived vesicles or tubules on the cis side of the stacks and then move successively toward the trans side as new cisternae are formed [60, 61]. Later, transport across the Golgi stacks was largely viewed as a discontinuous process in which carrier vesicles transfer material between stable subcompartments [62, 63]. However, it has become evident that the stacks are dynamic structures and probably less compartmentalized than pre-

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viously assumed. Thus, specific marker enzymes involved in glycoprotein synthesis and processing show an overlapping and variable distribution both within the stacks of a given cell type and when comparing the stacks of different cell types [64 – 66]. This has revived interest in the idea that the Golgi cisternae may be transient structures which move in cis to trans direction and carry proteins through the stacks. In parallel, vesicles or tubules may be responsible for retrograde transport of enzymes and other Golgi constituents [55, 67– 69]. Another model emerging from studies of living cells suggests that the Golgi stacks are linked together by tubular connections and that membrane components as well as luminal content may rapidly move between different parts of the stacks by lateral diffusion [70]. The close apposition of the cisternae in the central parts of the Golgi stacks has been attributed to the existence of fine elements linking adjacent cisternae together [61]. Studies of isolated Golgi fractions have indicated that these bridges are proteinaceous in nature, but their exact identity remains to be determined [71, 72]. One possibility is that resident Golgi proteins and proteins operating in vesicular transport between consecutive Golgi subcompartments hold the cisternae together in the stacked configuration [73–75]. N-Acetylglucosaminyltransferase I, an enzyme present in medial and trans cisternae and involved in the construction of N-linked oligosaccharides [76]; GM130, a cis-Golgi matrix protein [77, 78]; and GRASP65, a Golgi reassembly and stacking protein [79], are three examples of molecules that have been proposed to fulfill such a function. Intracellular Protein Transport Study of intracellular transport in eukaryotes from yeast to man has disclosed the existence of two general protein machineries used in the control of vesicle budding, targeting, and fusion [12, 80 – 82]. They involve a small GTP-binding protein (ARF or Sar1p) which binds to the membrane and recruits a complex of coat proteins (COPI or COPII), leading to the formation of a coated bud. A vesicle is then pinched off by periplasmic fusion, and after hydrolysis of the bound GTP the coat disassembles. With the help of integrated membrane proteins serving as specific address markers (SNAREs) and small GTP-binding proteins (Rab or Ypt) regulating the interaction between these molecules, the vesicle docks with its target membrane. A new set of proteins (NSF and SNAPs) available in the cytoplasm then binds to the site of contact. In an ATP-dependent process, these proteins mediate fusion of the attached membranes, completing one step in a chain of transport events. This mechanism has largely been worked out in studies on transport along the exocytic pathway,

i.e., between the ER and the Golgi complex and across the Golgi stacks. A similar system, including clathrin and adaptor complexes, functions in vesicle budding during endocytosis and packaging of lysosomal enzymes at the trans face of the Golgi stacks [83– 85]. Many details about the functions of the vesicle budding and fusion machineries mentioned above still wait to be resolved. It also needs to be further clarified which transport steps COPI and COPII take part in. The knowledge available today indicates that COPII-coated vesicles are obligate intermediates in the transport from the ER to the Golgi complex [86 –91]. With regard to COPI, it has been suggested that this coat may function both in transport from the ER–Golgi intermediate compartment to the Golgi stacks and in anterograde as well as retrograde transport through the stacks [59, 91–96]. MICROTUBULES AND THE ORGANIZATION OF THE GOLGI COMPLEX

Structural Relationship and Effects of Antimicrotubular Drugs As mentioned in the introduction, it has long been known that the structural integrity of the Golgi complex is dependent on microtubules [2]. Double-immunofluorescence microscopy has given a striking illustration of the close association of the Golgi elements with the center of the microtubule network in cultured cells and how the overall organization of Golgi is changed after drug-induced depolymerization of microtubules (Figs. 2A and B). Electron microscopy has in likewise manner been instrumental in demonstrating the morphological details of this process, i.e., the disconnection of the cisternal stacks and their dispersal in the cytoplasm in an altered configuration (Figs. 3A and B). Thus, the modification of the Golgi complex after microtubule disruption is less dramatic than that seen after treatment with other drugs [97, 98] such as brefeldin A, a fungal metabolite interfering with ARFdependent vesicle budding in the secretory pathway [99 –104]; okadaic acid, a protein phosphatase inhibitor [105–109]; and ilmaquinone, a drug acting via heterotrimeric G proteins [110 –112]. In the presence of these substances, the Golgi stacks are broken down into tubulovesicular clusters, and protein secretion ceases almost completely. On the other hand, the modified Golgi stacks of cells exposed to antimicrotubular drugs remain functional and protein secretion is only moderately affected [113–115]. However, in polarized epithelial cells transport to the apical surface is more dependent on microtubules than transport to the basolateral surface [116 –124]. Additional analysis revealed that the effect of antimicrotubular drugs on Golgi morphology can be di-

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FIG. 2. Double-immunofluorescence microscopy of cultured L929 mouse fibroblasts stained with primary antibodies against the Golgi marker enzyme mannosidase II and tubulin. Golgi elements appear in yellow and microtubules in green. (A) Control cells. (B) Cells treated with 10 mM nocodazole for 4 h. Bars mark 10 mm.

vided into three stages: microtubule depolymerization, fragmentation of the system of interconnected Golgi stacks, and fragment dispersal. Notably, microtubule depolymerization as such was not sufficient for the two latter events to take place. Metabolic energy and ongoing membrane traffic were also required [125]. More recently, it was demonstrated that the Golgi fragments are not randomly dispersed in the cytoplasm but redistributed close to ER exit sites. Accordingly, the intermediate compartment marker protein ERGIC-53 was found to rapidly relocate to these sites after microtubule disruption, whereas medial and trans Golgi marker enzymes (mannosidase II and galactosyltransferase) were moved to the same sites gradually over several hours [126]. Signs of separation of Golgi subcompartments from each other in the absence of microtubules were further obtained by the observation that trans Golgi proteins were scattered more rapidly than medial Golgi proteins during nocodazole treatment [127]. Summing up, these findings suggest that after

loss of microtubules, the Golgi stacks are disconnected and partly break up into smaller fragments. These move around in the cytoplasm and associate with subregions of the ER. The mechanisms behind the disconnection and breakup of the Golgi stacks and their subsequent movement and association with the ER remain unknown. Microtubule Subpopulations Microtubules rich in detyrosinated and/or acetylated tubulin have a longer half-life and are more resistant to drug-induced depolymerization than the majority of cytoplasmic microtubules and may be of special importance in the structural organization of cells [33, 34]. In studies on mouse L929 fibroblasts treated with nocodazole (3 mM), recovery in the presence of a low drug concentration (0.15 mM) was found to allow partial repolymerization of microtubules from the centrosomal region and an almost complete normalization of the

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FIG. 3. Electron micrographs demonstrating portions of the Golgi complex in cultured rat arterial smooth muscle cells kept in normal medium (A) or treated with 10 mM nocodazole for 4 h (B). In the control cell (A), the Golgi stacks (G) consist of flattened cisternae and are grouped together in the juxtanuclear region. In the nocodazole-treated cell (B), the Golgi stacks are dispersed in the cytoplasm and show an altered morphology with swollen cisternae and few surrounding vesicles. ER, endoplasmic reticulum; MT, microtubules. Bars mark 0.5 mm.

Golgi complex as judged fine structurally [128]. Subsequently, it was demonstrated by immunofluorescence microscopy that microtubules rich in detyrosinated and/or acetylated tubulin codistribute with the Golgi complex both under normal conditions and in cells recovering from nocodazole [129, 130]. Moreover, disruption of the Golgi complex with brefeldin A did not destabilize the associated network of detyrosinated microtubules and did not prevent the reformation of this network after exposure to nocodazole [131]. Immunoelectron microscopic analysis further disclosed a close connection between microtubules containing detyrosinated tubulin and vesicles engaged in the transport of newly synthesized secretory proteins from the ER to the Golgi complex [132]. These findings support the notion that stable microtubules, enriched in posttrans-

lationally modified tubulin, take part in the movement of material to and the organization of the Golgi complex. They also indicate that the existence of this subset of microtubules is not a result of the interaction with Golgi elements. Conceivably, long-lived microtubules function both as tracks for transport of Golgi elements toward the centrosomal region and as a framework to which Golgi elements may be anchored. Movement of Golgi Elements along Microtubules During the past 2 decades, a vast literature has accumulated to establish that microtubules function as roadways for mechanochemical motor proteins using the energy of ATP hydrolysis to transport membrane-bound organelles as well as other structures within the cell.

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Kinesin and cytoplasmic dynein have been identified as the two main microtubule-associated motors, kinesin usually serving in plus-end-directed transport and dynein in minus-end-directed transport [6–9]. As part of these efforts, microtubules have been shown to participate in the maintenance of the spatial organization of the ER and the Golgi complex and the routing of vesicular carriers to and from these organelles [133–136]. Recently, the actin-myosin system has also been implicated in membrane trafficking and it is possible that microtubules and actin filaments may cooperate in intracellular transport along the secretory pathway [137–140]. In a study on Vero fibroblasts treated with nocodazole, Ho et al. [141] demonstrated that, after removal of the drug, scattered Golgi elements moved along reassembling microtubules back to the centrosomal region. The individual Golgi complexes of fused cells were likewise found to move along microtubules to the center of the newly formed syncytia and unite into a large extended Golgi complex within a few hours [142]. By fluorescence imaging of living astrocytes stained with NBD–ceramide and subsequent visualization of microtubules in the same cells by immunofluorescence microscopy, trans-Golgi cisternae were shown to form processes that extend along microtubules and link adjacent Golgi stacks together in a continuous network. During treatment with nocodazole, no such links formed and the network was broken up into multiple fragments [143]. As mentioned earlier, the Golgi complex is typically localized in the region around the centrosome, where the minus end of microtubules is anchored in fibroblasts and several other cell types (a different situation may exist in polarized epithelial cells). Cytoplasmic dynein was therefore considered a candidate motor protein taking part in the organization of the Golgi complex. Experiments on semi-intact CHO cells revealed that isolated Golgi elements interact with microtubules and collect near the centrosome in a dyneindependent manner [144]. Both dynein and kinesin have further been found to bind to Golgi membranes [145–150]. In support of a role of these proteins in membrane trafficking, microinjection of dynein heavy chain antibodies was shown to disperse the Golgi complex of NRK cells [149]. Recently, it was also demonstrated that the Golgi complex of mouse cells lacking cytoplasmic dynein is fragmented and widely spread out in the cytoplasm [151]. In analogy, microinjection of kinesin antibodies was observed to inhibit Golgito-ER transport in NRK cells treated with brefeldin A [147] and the scattering of Golgi elements along remaining microtubules in human fibroblasts exposed to a low concentration (0.1 mM) of nocodazole [152]. On the other hand, no interference with the brefeldin Ainduced breakdown of the Golgi complex was noted in mouse cells with a targeted disruption of the ubiquitous kinesin heavy chain [153].

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FIG. 4. A schematic view of the role of the microtubule motor proteins cytoplasmic dynein and kinesin in membrane transport to and from the Golgi complex. The plus and minus ends of microtubules are indicated. C, centrosome; ER, endoplasmic reticulum; MT, microtubules; N, nucleus; PM, plasma membrane.

Taken together, these results strongly implicate microtubules and microtubule motors in the arrangement and function of the Golgi complex (Fig. 4). At least in fibroblastic cells, the Golgi stacks are positioned in the region around the centrosome, close to the origin of the radiating system of cytoplasmic microtubules. Cytoplasmic dynein supports the microtubule-dependent trafficking of newly produced membrane and cargo proteins from the ER to the cis side of the Golgi stacks as well as the recycling of membrane from the cell surface to the trans side of the stacks (via endosomes). In analogy, kinesin may support transport of resident ER components back from the cis side of the Golgi stacks and movement of membrane and secretory proteins from the trans side of the stacks to the cell surface. Microtubules also aid to connect the Golgi stacks into an uninterrupted network. However, the molecular mechanisms in the interaction between microtubules and membranes are incompletely understood. A large variety of proteins capable of modulating the binding of microtubule motors to membranes have been identified, including dynamin [154 –156], kinectin [157, 158], and spectrin [159, 160]. Moreover, myosin has been observed to associate with Golgi membranes, suggesting a role also for actin/myosin filaments in vesicular traffic to and from this organelle [145, 161, 162]. To establish how microtubules and actin/myosin filaments act together in biogenesis and maintenance of the Golgi complex remains a challenge for the future. REORGANIZATION OF THE GOLGI COMPLEX DURING MITOSIS

As pointed out in our earlier review [2], the Golgi complex breaks down into small, widely dispersed frag-

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Prophase

FIG. 5. A schematic model of the reorganization of the Golgi complex during mitosis. In interphase, an interconnected system of cisternal stacks is found around the centrosome, in close contact with the radiating array of cytoplasmic microtubules. In prophase, microtubules depolymerize and the Golgi stacks are disengaged from each other and dispersed. In metaphase and anaphase, the stacks are further fragmented and small clusters of vesicular and tubular elements appear throughout the cytoplasm. In telophase, the Golgi stacks are reformed and gather close to the intercellular bridge. At the end of cytokinesis, when the daughter cells are about to move apart, the Golgi stacks are translocated to the other side of the nucleus. During this reassembly and repositioning process, the Golgi stacks remain closely related to the centrosome and the reforming system of cytoplasmic microtubules.

ments during mitosis and this is the basis for its partitioning between the daughter cells (a similar modification is also seen in the ER). A schematic model of this process is given in Fig. 5. At the same time, membrane transport along the endocytic and exocytic pathways is inhibited [163–165]. These changes are due partly to the disruption of the interphase array of cytoplasmic microtubules and partly to an altered activity of proteins involved in the fusion of vesicles with their target membranes. Mitosis-promoting factor (MPF), a complex of cdc2 kinase and cyclin B, is a key regulator of these and other events in the G 2–M transition of the cell cycle [166, 167].

During prophase, cytoplasmic microtubules disassemble as a result of endogenous calcium transients [168], changes in the nucleating activity of the centrosomes [18], and modulation of microtubule dynamics [169, 170]. Using fluorescence ratio and fluorescence photoactivation methods, it was shown that an abrupt decrease in microtubule polymer level and an increase in microtubule dynamics occur at the time of nuclear envelope breakdown [171]. Three proteins that may take part in this rapid destruction of interphase microtubules have been described, p56 [172], elongation factor 1a [173], and katanin [174]. Katanin is an ATPase concentrated at the centrosomes throughout the cell cycle [175–177]. Like the other proteins mentioned above, its microtubule-severing activity is apparently under control of cdc2 kinase, but the mechanism is not known [169]. Anyway, it was previously demonstrated that microinjection of cdc2 kinase induces a marked reduction in interphase microtubules [178]. Using cellfree extracts, it was further shown that cdc2 kinase causes a shift in microtubule dynamics (increased catastrophe frequency) similar to that noted at the onset of mitosis [179, 180]. This effect may in part be mediated via phosphorylation of microtubule-associated proteins such as MAP4 [181]. It must be stressed, however, that removal of MAP4 from microtubules in vivo did not give any clear phenotype at the cellular level, suggesting that a functional redundancy exists between different MAPs [182]. Another family of protein kinases, MARK, that phosphorylate MAPs and trigger microtubule disruption has also been described [183, 184], but the role of these enzymes in mitosis has not yet been defined. During prophase, the Golgi complex likewise begins to break up [163–165]. The cisternal stacks become smaller, disconnect from each other, and adopt a perinuclear and more widely dispersed distribution [185– 187]. These changes are analogous to those seen in interphase cells treated with microtubule-disruptive drugs (see above), and it seems likely that they to a large extent are a result of the aforementioned loss of cytoplasmic microtubules. In the absence of these tracks, the directed transport of Golgi elements toward the centrosomal region will cease, making it difficult to keep the cisternal stacks together in an uninterrupted network [141, 143]. Moreover, the ability of the motor protein cytoplasmic dynein to associate with membranes was found to decrease during mitosis. This may prevent vesicle binding to the spindle, which starts to form late in prophase [188, 189]. The morphology and function of the Golgi complex in mitotic cells are strongly influenced by cdc2 and other protein kinases also in a more direct manner (independent of the effects on microtubule integrity). However, in this case

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the effects become manifest somewhat later and therefore will be described below. Metaphase and Anaphase Early in metaphase, the nuclear envelope is disrupted and its integral membrane proteins are dispersed throughout the ER [190]. In addition, the ER itself is broken up into small vesicular and tubular elements [165]. This enables the spindle microtubules to establish contact with the chromosomes and eventually align them in the metaphase plate. At this time, a more extensive fragmentation of the Golgi stacks is also noted, generating numerous clusters of small vesicular and tubular profiles [185–187, 191–194]. In HeLa cells, the clusters were found to contain cis- as well as trans-Golgi markers, indicating that the different parts of the stacks remain closely associated after completion of the disassembly process [187, 191, 193– 195]. On the other hand, studies on L929 and CHO cells suggested that cis and medial parts of the stacks at least partially fuse with the ER (like in cells treated with brefeldin A) and that the clusters primarily originate from the trans parts of the stacks [186]. As referred to above, it was similarly found that Golgi subcompartments are capable of separating from each other in cells treated with antimicrotubular drugs [126, 127]. In parallel with the structural breakdown of the Golgi complex, transport of membrane and luminal proteins along the secretory pathway is inhibited, with a major block in ER-to-Golgi transfer [196 –198]. Concerning newly synthesized membrane lipids, more variable results were obtained, possibly on account of the existence of alternative routes for delivery of lipids to Golgi and the cell surface [199, 200]. Using a cellfree system, evidence has accumulated to support the notion that the mitotic fragmentation of the Golgi stacks is caused by continued budding of transport vesicles by a combination of COP-dependent and COPindependent mechanisms and a concomitant inhibition of vesicle fusion with the target membranes [201–203]. It was later disclosed that the arrest of intra-Golgi transport is due to an inability of the vesicle docking protein p115 to bind to Golgi membranes under mitotic conditions, obviously as a result of phosphorylation of GM130, the membrane-bound receptor for p115 [78, 204, 205]. Cyclin-dependent kinases are likely to be involved in this phosphorylation and, notably, cyclin B2 (a cyclin that associates with and activates cdc2 kinase during mitosis) was shown to be localized to the Golgi complex [206]. In contrast, a recent study on permeabilized NRK cells revealed that Golgi fragmentation by mitotic extracts did not require the continued presence of cdc2 kinase. Instead, it depended on one of the kinases in the mitogen-activated protein kinase

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cascade, MEK1. The normal cytoplasmic substrates of MEK1, ERK1 and ERK2, were not involved, however, perhaps indicating that Golgi membranes hold an ERK-like activity [207]. The relation between cdc2 kinase and MEK1 in the signaling pathway for Golgi fragmentation awaits to be settled. Transport of material into the cell by endocytosis is also suppressed during mitosis [208, 209], apparently as a result of inhibition both of receptor recycling [210, 211] and invagination of clathrin-coated pits [212, 213]. It was further demonstrated that endocytic vesicle fusion in a cell-free system was reduced if using mitotic rather than interphase cytosol, if adding cdc2 kinase to interphase cytosol (together with cyclin A or B), or if including protein phosphatase inhibitors such as okadaic acid and microcysin-LR in the incubations [214 –216]. In addition, studies on CHO cells have revealed that endosomes and lysosomes (labeled by prior exposure of the cultures to horseradish peroxidase) markedly decrease in size during metaphase and anaphase and at the same time increase in number [165]. These findings may be explained by continued vesicle budding and concurrent cessation of vesicle fusion. They agree with the idea that ingested material is normally spread within the endosomal–lysosomal system and mixed with acid hydrolases by continuous fission and fusion processes [83]. Summing up, notable similarities exist in the manner in which the Golgi complex and the endosomes–lysosomes are fragmented during mitosis. Telophase and Cytokinesis As mentioned in the beginning of this section, the complex of cdc2 kinase and cyclin B (MPF) is essential for the initiation of mitosis and appears to be involved directly or indirectly in most of the changes described above [166, 167]. However, once the chromosomes have been aligned in the metaphase plate, cyclin B and other proteins must be degraded in order to allow the cell first to progress into anaphase (sister chromatid separation) and later to exit telophase. This is accomplished by activation of the anaphase-promoting complex, a multisubunit complex that catalyzes the synthesis of polyubiquitin chains on several proteins (including mitotic cyclins), thereby targeting them for destruction by proteasomes [217, 218]. As a result, the morphological reorganization of the cell can be reversed. Thus, a new radiating system of cytoplasmic microtubules as well as a normalized ER and Golgi complex start to reform in the daughter cells before they detach from each other in cytokinesis. With regard to the Golgi complex, the reassembly of the dispersed vesicular and tubular elements into cisternal stacks grouped together in a defined region of the cytoplasm has been found to proceed in the pres-

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ence of RNA and protein synthesis inhibitors [188, 219]. It was also shown to occur before newly synthesized proteins arrive from the ER [220]. Hence, the proteins needed to reconstruct Golgi stacks in the daughter cells are reutilized and do not have to be produced de novo. Electron microscopic studies and studies of living cells have revealed that at least two steps are involved: first Golgi clusters grow larger by accumulation of free vesicles and later cisternae form by membrane fusion within the clusters [193, 221]. In parallel, a close relationship to the centrosome and the microtubular cytoskeleton is reestablished [186], presumably by transport of Golgi elements along reforming microtubules in a manner similar to that in cells recovering from treatment with microtubule-disruptive drugs [141]. The reappearance of stable, posttranslationally modified microtubules may also be important here [130]. Reassembly of Golgi stacks from mitotic fragments has been examined in further detail using a cell-free system [222]. The results denote that this process requires two ATPases, NSF and p97 [223, 224], as well as the SNARE protein syntaxin 5 [225]. It therefore seems as if the rebuilding of Golgi stacks at the end of mitosis makes use of a machinery analogous to that employed for membrane fusion during intraGolgi transport in interphase [12, 82]. Studies of cells at final stages of division have demonstrated that the centrosomes and the Golgi complex go through a synchronized change in location following karyokinesis and before completion of cytokinesis [226, 227]. Initially, they relocate from the poles to the region adjacent to the intercellular bridge (on the other side of the nucleus). Later, preceding the final separation of the daughter cells, the centrosome and the Golgi complex move back to the poles. Immunocytochemical analyses on rat embryo fibroblasts have indicated that this shift in Golgi localization is accompanied by a shift in the directed transport of secretory and membrane proteins (such as fibronectin and fibronectin receptors) to the cell surface [228]. Based on these observations, it was suggested that the change in position of the Golgi complex in association with cytokinesis serves the function of directing transport of secretory and membrane proteins to different parts of the cell surface at different times. This could help the daughter cells first to establish contact with each other and then to lay down a substrate on which they spread out and move apart. The regulatory mechanisms behind these intricate changes in internal organization and polarized behavior of cells at the end of mitosis are not known. CONCLUDING REMARKS

The Golgi complex of mammalian cells is made up of cisternal stacks that function in processing and sorting of proteins en route from the endoplasmic reticulum to

lysosomes, secretory vacuoles, and the cell surface. The stacks are usually arranged as an interconnected network in the region around the centrosome, the main organizing center for cytoplasmic microtubules. Two factors are of primary importance for the integrity of Golgi. One is the budding and fusion of vesicles carrying membrane and cargo from the ER to the cis side of the stacks, through the latter, and from the their trans side to various destinations. The sorting mechanisms built into this machinery give Golgi its characteristic composition and ensure the specificity of transport along different pathways. Another key factor in the construction of the Golgi complex is the interaction of membranes with cytoplasmic microtubules. With the help of motor proteins such as cytoplasmic dynein and kinesin, microtubules serve as tracks along which vesicles move to and from the Golgi. Likewise, the Golgi elements themselves move along microtubules and cluster close to their minus ends. The phosphorylationdependent regulation of the aforementioned machineries for membrane budding–fusion and membrane–microtubule interaction enables the cell not only to uphold the normal interphase morphology of the Golgi but also to break up this single-copy organelle during mitosis and divide it equally between the daughter cells. In evolutionarily distant species like insects, plants, and fungi, the organization of the Golgi complex is different from that in higher animal cells. For example, Golgi in Chironomus and Drosophila cells are found as discrete small stacks dispersed in the cytoplasm and without any clear spatial relationship to microtubules [229, 230]. A similar pattern with scattered cisternal stacks (dictyosomes) is noted in plant cells [61, 231]. Stacking of Golgi cisternae is also evident in fission yeast [232], whereas Golgi of budding yeast consist of single, isolated cisternae [233, 234]. Nevertheless, the absence of microtubules was observed to affect Golgi morphology in both cell types. In fission yeast, treatment with the antimicrotubular agent thiabendazole caused unstacking of Golgi cisternae [232]. In mutants of budding yeast lacking b-tubulin, membrane tubules believed to represent Golgi were broken down into smaller fragments [234]. Even if the mechanism may be different, it thus seems as if microtubules take part in the assembly of the Golgi complex also in yeast. From a developmental point of view, the arrangement of the Golgi complex as a unified system in the pericentrosomal region of mammalian cells may be seen as a means of bringing specific and closely related functions together in defined subcompartments and with defined transport routes for incoming and outgoing material. This is likely to be significant in order to support an effective functioning of the multitude of biosynthetic and catabolic pathways distinguishing these cells.

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The original work described in this report was supported by grants from the Swedish Medical Research Council, the Swedish Heart Lung Foundation, the King Gustaf V 80th Birthday Fund, and Karolinska Institutet.

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