Who Needs Microtubules? Myogenic Reorganization of MTOC, Golgi Complex and ER Exit Sites Persists Despite Lack of Normal Microtubule Tracks
Kristien J.M. ZaalEricka ReidKambiz MousaviTan ZhangAmisha MehtaElisabeth BugnardVittorio SartorelliEvelyn Ralston
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A wave of structural reorganization involving centrosomes, microtubules, Golgi complex and ER exit sites takes place early during skeletal muscle differentiation and completely remodels the secretory pathway. The mechanism of these changes and their functional implications are still poorly understood, in large part because all changes occur seemingly simultaneously. In an effort to uncouple the reorganizations, we have used taxol, nocodazole, and the specific GSK3-β inhibitor DW12, to disrupt the dynamic microtubule network of differentiating cultures of the mouse skeletal muscle cell line C2. Despite strong effects on microtubules, cell shape and cell fusion, none of the treatments prevented early differentiation. Redistribution of centrosomal proteins, conditional on differentiation, was in fact increased by taxol and nocodazole and normal in DW12. Redistributions of Golgi complex and ER exit sites were incomplete but remained tightly linked under all circumstances, and conditional on centrosomal reorganization. We were therefore able to uncouple microtubule reorganization from the other events and to determine that centrosomal proteins lead the reorganization hierarchy. In addition, we have gained new insight into structural and functional aspects of the reorganization of microtubule nucleation during myogenesis.Keywords:
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Centrosomes direct the organization of microtubules in animal cells. However, in the absence of centrosomes, cytoplasm has the potential to organize microtubules and assemble complex structures such as anastral spindles. During cell replication or following fertilization, centrioles that are incapable of organizing microtubules into astral arrays are introduced into this complex cytoplasmic environment. These centrioles become associated with pericentriolar material responsible for centrosome‐dependent microtubule nucleation, and thus the centrosome matures to ultimately become a dominant microtubule organizing center that serves as a central organizer of cell cytoplasm. We describe the identification of a novel structure within the pericentriolar material of centrosomes called the centromatrix. The centromatrix is a salt‐insoluble filamentous scaffold to which subunit structures that are necessary for microtubule nucleation and abundant in the cytoplasm bind. We propose that the centromatrix serves to concentrate and focus these subunits to form the microtubule organizing center. Since binding of these subunits to the centromatrix does not require nucleotides, we propose a model for centrosome assembly which predicts that the assembly of the centromatrix is a rate‐limiting step in centrosome assembly and maturation.
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Although the centrosome is traditionally viewed as cell’s principle microtubule organizing center (MTOC), regulation of microtubule dynamics at the cell cortex plays an equally important role in the formation of the steady-state microtubule network. Several recent studies, including one published in this issue, reveal that complex signaling mechanisms associated with adherence junctions influence both microtubule nucleation at the centrosome, and the stability of non-centrosomal microtubules. In the mid 1980s Marc Kirschner and Timothy Mitchison proposed an elegant “search-and-capture” hypothesis that seemed to explain how cells manage to convert a simple radial array of microtubules produced by the centrosome into the complex and precisely regulated asymmetric network found in a typical polarized cell. The key to this mechanism was the selective stabilization of inherently dynamic microtubule plus ends at the certain parts of cell cortex.4 Subsequently, it was shown that microtubule plus ends can in fact be captured and stabilized at diverse cortical loci including focal adhesions and adherence junctions. These observations provided direct support to the search-and-capture hypothesis. However, in recent years it became clear that role of cell cortex in the regulation of microtubule dynamics goes beyond simple stabilization of the plus ends. For example, there is evidence that integrin β1 is involved in the regulation of microtubule nucleation at the centrosome.6 Further, in polarized epithelia, cell cortex serves as the dominant MTOC, effectively replacing the centrosome.5 Thus, cell-cortex mechanisms affect microtubule dynamics both at their plus- and minus ends. The challenge now is to identify molecular pathways underlying this regulation. A study in this issue of Cell Cycle (Shtutman et al.) suggests that α-catenin, a major component of adherence junctions is responsible for promoting microtubule nucleation and/or stability in a centrosome-independent fashion. Shtutman and coworkers used centrosome-free cytoplasts. The number of microtubules in these cytoplasts is low in the absence of cell-cell contacts but increases to near-normal levels in confluent cultures3 or upon overexpression of cadherins1 suggesting that adherence junctions somehow regulate microtubule dynamics. Shtutman and coworkers now demonstrate a similar increase in microtubule density can be induced by overexpression of a membrane-targeted α catenin. This is an exciting finding because α-catenin is also directly involved in the regulation of actin dynamics2 and thus this molecule emerges as a central player in the global regulation of the cytoskeleton in response to extracellular interactions. Interestingly, expression of non-membrane-targeted α-catenin only mildly increased the density of microtubule network in centrosome-free cytoplasts suggesting that α-catenin needs to be engaged in an activation event at the cell cortex, perhaps within the adherence junction. Although formation of cell-cell junctions clearly increases the density of microtubule network, microtubule nucleation appears to occur throughout the cytoplasm and not preferentially at adherence junctions in these cells.1 Thus, local interactions at adherence junctions ultimately result in the propagation of a certain factor(s) that influences global microtubule dynamics. The exact nature of this factor or even the general layout of the pathway that alters microtubule dynamics in response to cortical interactions remain unknown. However, the demonstration that α-catenin is one of the molecular players required for this pathway is an important towards the understanding the link between extracellular interactions and microtubule dynamics. Further ReadingChausovsky A, Bershadsky AD, Borisy GG. Cadherin-mediated regulation of microtubule dynamics. Nat Cell Biol 2000; 2:797- 804. Gates J, Peifer M. Can 1000 reviews be wrong? Actin, alpha-Catenin, and adherens junctions. Cell 2005; 123:769-72. Karsenti E, Kobayashi S, Mitchison T, Kirschner M. Role of the centrosome in organizing the interphase microtubule array: properties of cytoplasts containing or lacking centrosomes. J Cell Biol 1984; 98:1763-76. Kirschner M, Mitchison T. Beyond self-assembly: from microtubules to morphogenesis. Cell 1986; 45:329-42. Reilein A, Yamada S, Nelson WJ. Self-organization of an acentrosomal microtubule network at the basal cortex of polarized epithelial cells. J Cell Biol 2005; 171:845-55. Reverte CG, Benware A, Jones CW, LaFlamme SE. Perturbing integrin function inhibits microtubule growth from centrosomes, spindle assembly, and cytokinesis. J Cell Biol 2006; 174:491-7.
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Control of microtubule nucleation is important for many microtubule dependent processes in cells. Traditionally, research has focused on nucleation of microtubules from centrosomes. However, it is clear that microtubules can nucleate from non-centrosome dependent sites. In this review we discuss the consequences of non-centrosome dependent microtubule nucleation for formation of microtubule patterns, concentrating on the assembly of mitotic spindles.
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ABSTRACT Control of microtubule nucleation is important for many microtubule dependent processes in cells. Traditionally, research has focused on nucleation of microtubules from centrosomes. However, it is clear that microtubules can nucleate from non-centrosome dependent sites. In this review we discuss the consequences of non-centrosome dependent microtubule nucleation for formation of microtubule patterns, concentrating on the assembly of mitotic spindles.
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Microtubule organization and nucleation were studied during in vitro human myogenesis by immunocytology that used monoclonal and polyclonal antitubulin antibodies and a rabbit nonimmune serum that reacts with human centrosomes. In myoblasts, we observed a classical microtubule network centered on juxtanuclear centrosomes. Myotubes possessed numerous microtubules organized in parallel without any apparent nucleation centers. Centrosomes in these cells were not associated one to each nucleus but were often clustered in the vicinity of nuclei groups. They were significantly smaller than those of the mononucleated cells. The periphery of each nucleus in myotubes was labeled with the serum that labels centrosomes suggesting a profound reorganization of microtubule-nucleating material. Regrowth experiments after Nocodazole treatment established that microtubules were growing from the periphery of the nuclei. The redistribution of nucleating material was shown to take place early after myoblast fusion. Such a phenomenon appears to be specific to myogenic differentiation in that artificially induced polykaryons behaved differently: the centrosomes aggregated to form only one or a few giant nucleating centers and the nuclei did not participate directly in the nucleation of microtubules. The significance of these results is discussed in relation to the possible role of the centrosome in establishing cell polarity.
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The centrosome must be replicated once, and only once, during each cell cycle. To achieve this somatic cells need to synthesize centrosome proteins, target those centrosome proteins to the parental centrosome, and then assemble the centrosome subunits into a functional organelle. The mechanisms that underlie each of these processes are not known. Studies were performed to investigate whether cellular microtubules are involved in centrosome doubling events. For these experiments, CHO cells were arrested in either hydroxyurea (HU) alone or in HU plus a microtubule inhibitor for 36–40 h. The cells then were induced to enter mitosis and the numbers of spindle poles/centrosomes were counted following processing of the cells for immunofluorescence microscopy using anticentrosome antiserum. These studies demonstrated that centrosome replication events occurred in cells arrested with either HU alone or HU and taxol while centrosome replication did not occur in cells treated with HU and either nocodazole or colcemid. Immunoblot analysis determined that centrosome proteins were synthesized in HU/nocodazole-arrested cells and demonstrated that the role of microtubules in the centrosome replication process is not to ensure the synthesis of centrosome subunits. Rather, our results suggest that microtubules may be involved in the transport/targeting of centrosome subunits to the parental centrosome during duplication events. For microtubules to contribute to the transport of centrosome subunits during centrosome doubling, centrosome subunits would need to be able to bind to microtubules. To test this, co-sedimentation studies were performed and it was determined that the centrosome proteins, though overproduced under these conditions, remained soluble in HU/nocodazole-treated cells and co-pelleted with taxol-stabilized microtubules in the presence of GTP and AMP-PNP. Moreover, co-sedimentation of one of the centrosome proteins, PCM-1, with microtubules could be inhibited by pre-incubation of extracts with antibodies against dynactin. Together, these data suggest that during centrosome replication in somatic mammalian cells, PCM-1, and perhaps other centrosome components, are targeted to the centrosome via transport along microtubules by motor complexes that include dynein/dynactin. Cell Motil. Cytoskeleton 42:60–72, 1999. © 1999 Wiley-Liss, Inc.
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ABSTRACT In this work, we have investigated in mammalian cells how microtubule nucleation at centrosome and Golgi apparatus are coordinated, using genetic ablation of three γ-TuRC binding proteins -AKAP450, Pericentrin and CDK5Rap2- and the PLK4 inhibitor centrinone. We show that centrosomal microtubule nucleation is independent of Golgi activity whereas the converse is not true: nucleation on the Golgi negatively correlates with the number of centrosomes. In addition, depleting AKAP450 in cells lacking centrioles, that abolishes Golgi nucleation activity, leads to microtubule nucleation from numerous cytoplasmic Golgi-unbound acentriolar structures containing Pericentrin, CDK5Rap2 and y-tubulin. Strikingly, centrosome-less cells display twice higher microtubule density than normal cells, suggesting that the centrosome controls the spatial distribution of microtubules, not only by nucleating them, but also by acting as a negative regulator of alternative MTOCs. Collectively, the data reveals a hierarchical control of microtubule nucleation, with the centrosome regulating this process in a more complex manner than usually thought. It also unveils mechanisms that could help understanding MT network reorganization during cell differentiation.
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