The centrosome–Golgi apparatus nexus

A shared feature among all microtubule (MT)-dependent processes is the requirement for MTs to be organized in arrays of defined geometry. At a fundamental level, this is achieved by precisely controlling the timing and localization of the nucleation events that give rise to new MTs. To this end, MT nucleation is restricted to specific subcellular sites called MT-organizing centres. The primary MT-organizing centre in proliferating animal cells is the centrosome. However, the discovery of MT nucleation capacity of the Golgi apparatus (GA) has substantially changed our understanding of MT network organization in interphase cells. Interestingly, MT nucleation at the Golgi apparently relies on multiprotein complexes, similar to those present at the centrosome, that assemble at the cis-face of the organelle. In this process, AKAP450 plays a central role, acting as a scaffold to recruit other centrosomal proteins important for MT generation. MT arrays derived from either the centrosome or the GA differ in their geometry, probably reflecting their different, yet complementary, functions. Here, I review our current understanding of the molecular mechanisms involved in MT nucleation at the GA and how Golgi- and centrosome-based MT arrays work in concert to ensure the formation of a pericentrosomal polarized continuous Golgi ribbon structure, a critical feature for cell polarity in mammalian cells. In addition, I comment on the important role of the Golgi-nucleated MTs in organizing specialized MT arrays that serve specific functions in terminally differentiated cells.     

Centrosomal and non-centrosomal microtubules.

While microtubule (MT) arrays in cells are often focused at the centrosome, a variety of cell types contain a substantial number of non-centrosomal MTs. Epithelial cells, neurons, and muscle cells all contain arrays of non-centrosomal MTs that are critical for these cells' specialized functions. There are several routes by which non-centrosomal MTs can arise, including release from the centrosome, cytoplasmic assembly, breakage or severing, and stabilization from non-centrosomal sites. Once formed, MTs that are not tethered to the centrosome must be organized, which can be accomplished by means of self-organization or by capture and nucleation of MTs where they are needed. The presence of free MTs requires stabilization of minus ends, either by MT-associated proteins or by an end-capping complex. Although some of the basic elements of free MT formation and organization are beginning to be understood, a great deal of work is still necessary before we have a complete picture of how non-centrosomal MT arrays are assembled in specific cell types.     

Cytoplasmic dynein is involved in the retention of microtubules at the centrosome in interphase cells

Cytoplasmic dynein is known to be involved in the establishment of radial microtubule (MT) arrays. During mitosis, dynein activity is required for tethering of the MTs at the spindle poles. In interphase cells, dynein inhibitors induce loss of radial MT organization; however, the exact role of dynein in the maintenance of MT arrays is unclear. Here, we examined the effect of dynein inhibitors on MT distribution and the centrosome protein composition in cultured fibroblasts. We found that while these inhibitors induced rapid ( t _1/2 ∼ 20 min) loss of radial MT organization, the levels of key centrosomal proteins or the rates of MT nucleation did not change significantly in dynein-inhibited cells, suggesting that the loss of dynein activity does not affect the structural integrity of the centrosome or its capacity to nucleate MTs. Live observations of the centrosomal activity showed that dynein inhibition enhanced the detachment of MTs from the centrosome. We conclude that the primary role of dynein in the maintenance of a radial MT array in interphase cells consists of retention of MTs at the centrosome and hypothesize that dynein has a role in the MT retention, separate from the delivery to the centrosome of MT-anchoring proteins.     

Microtubule organization during development of stomatal complexes inLolium rigidum

Summary Microtubule (MT) arrays in stomatal complexes ofLolium have been studied using cryosectioning and immunofluorescence microscopy. This in situ analysis reveals that the arrangement of MTs in pairs of guard cells (GCs) or subsidiary cells (SCs) within a complex is very similar, indicating that MT deployment is closely coordinated during development. In premitotic guard mother cells (GMCs), MTs of the transverse interphase MT band (IMB) are reorganized into a longitudinal array via a transitory array in which the MTs appear to radiate from the cell edges towards the centre of the walls. Following the longitudinal division of GMCs, cortical MTs are reinstated in the GCs at the edge of the periclinal and ventral walls. The MTs become organized into arrays which radiate across the periclinal walls, initially from along the length of the ventral wall and later only from the pore site. As the GCs elongate, the organization of MTs and the patterns of wall expansion differ on the internal and external periclinal walls. A final reorientation of MTs from transverse to longitudinal is associated with the elongation and constriction of GCs to produce mature complexes. During cytokinesis in the subsidiary mother cells (SMCs), MTs appear around the reforming nucleus in the daughter epidermal cells but appear in the cortex of the SC once division is complete. Our results are thus consistent with the idea that interphase MTs are nucleated in the cell cortex in all cells of the stomatal complex but not in adjacent epidermal cells.Abbreviations GMC guard mother cell - GC guard cell - IMB interphase microtubule band - MT microtubule - PPB preprophase band - SMC subsidiary mother cell - SC subsidiary cell     

Processes induced by tau expression in Sf9 cells have an axon-like microtubule organization

We have indirectly analyzed the role of tau in generating the highly organized microtubule (MT) array of the axon. Axons contain MT arrays of uniform polarity orientation, plus ends distal to the cell body (Heidemann, S. R., J. M. Landers, and M. A. Hamborg. 1981. J. Cell Biol. 91:661-673). Surprisingly, these MTs do not radiate from a single discrete nucleating structure in the cell body (Sharp, G. A., K. Weber, and M. Osborn. 1982. Eur. J. Cell Biol. 29: 97-103), but rather stop and start at multiple sites along the length of the axon (Bray, D., and M. B. Bunge. 1981. J. Neurocytol. 10:589-605). When Sf9 ovarian cells are induced to express high levels of tau protein, they develop cellular processes which are similar in appearance to axons and which contain dense arrays of MTs (Knops, J., K. S. Kosik, G. Lee, J. D. Pardee, L. Cohen-Gould, and L. McConlogue. 1991. J. Cell Biol. 114:725-734). We have analyzed the organization of MTs within these arrays, and determined it to be similar, but not identical, to the organization of MTs within the axon. The caliber, MT number, and MT density vary significantly from process to process, but on average are manyfold higher in the tau-induced processes than typically found in axons. Greater than 89% of the MTs in the processes are oriented with their plus ends distal to the cell body, and this proportion is even higher in the processes that are most similar to axons with regard to caliber, MT number, and MT density. Similar to the situation in the axon, MTs are discontinuous along the length of the tau-induced processes, and do not emanate from any observable nucleating structure in the cell body. We have also identified bundles of MTs throughout the cell bodies of the Sf9 cells induced to express tau. Similar to the MT arrays in the processes, these MT bundles are not visibly associated with any other cytological structures that might regulate their polarity orientation. Nevertheless, these bundles consist of MTs most (greater than 82%) of which have the same polarity orientation. Collectively, these results suggest that tau may play a fundamental role in generating MT organization in the axon. In particular, a key property of tau may be to bundle MTs preferentially with the same polarity orientation.     

Arabidopsis GCP2 and GCP3 are part of a soluble gamma-tubulin complex and have nuclear envelope targeting domains

In higher plants, microtubules (MTs) are assembled in distinctive arrays in the absence of a defined organizing center. Three MT nucleation sites have been described: the nuclear surface, the cell cortex and cortical MT branch points. The Arabidopsis thaliana (At) genome contains putative orthologues encoding all the components of characterized mammalian nucleation complexes: gamma-tubulin and gamma-tubulin complex proteins GCP2 to GCP6. We have cloned the cDNA encoding AtGCP2, and show that gamma-tubulin, AtGCP2 and AtGCP3 are part of the same tandem affinity-purified complex and are present in a large membrane-associated complex. In addition, small soluble gamma-tubulin complexes of the size expected for a gamma-tubulin core complex are recruited to isolated nuclei. Using immunogold labelling, AtGCP3 is localized to both the nuclear envelope (NE) and the plasma membrane. To identify domains that could play a role in targeting complexes to these nucleation sites, truncated AtGCP2- and AtGCP3-green fluorescent protein fusion proteins were expressed in BY-2 cells. Several domains from AtGCP2 and AtGCP3 are capable of targeting fusions to the NE. We propose that regulated recruitment of soluble gamma-tubulin-containing complexes is responsible for nucleation at dispersed sites in plant cells and contributes to the formation and organization of the various MT arrays.     

Nanoscale and geometric influences on the microtubule cytoskeleton in plants: thinking inside and outside the box

The dynamic microtubule (MT) cytoskeleton found in the cell cortex of plants drives cell expansion via cell wall modifications. In the last decade, live cell imaging studies employing green fluorescent protein have helped unravel the mechanisms behind how cells arrange cortical MTs into complex arrays and shape cell expansion. In this review, we explore the reverse scenario: how cell geometry and organelles influence and constrain the organization and behavior of cortical MTs. This newly emerging principle explains how cells perceive local nanoscale structural input from MT-organizing centers, such as the nucleus, endomembranes, and cell edges, and translate this into global cell-wide order via MT self-organization. Studies primarily using the model plant Arabidopsis thaliana and tobacco BY-2 suspension cultures have broadened our understanding of how cells form not only elegant parallel arrays but also more complex MT configurations, including the prominent MT bundles found in preprophase bands, leaf epidermal cells, and developing xylem.     

Nanoscale and geometric influences on the microtubule cytoskeleton in plants: thinking inside and outside the box

The dynamic microtubule (MT) cytoskeleton found in the cell cortex of plants drives cell expansion via cell wall modifications. In the last decade, live cell imaging studies employing green fluorescent protein have helped unravel the mechanisms behind how cells arrange cortical MTs into complex arrays and shape cell expansion. In this review, we explore the reverse scenario: how cell geometry and organelles influence and constrain the organization and behavior of cortical MTs. This newly emerging principle explains how cells perceive local nanoscale structural input from MT-organizing centers, such as the nucleus, endomembranes, and cell edges, and translate this into global cell-wide order via MT self-organization. Studies primarily using the model plant Arabidopsis thaliana and tobacco BY-2 suspension cultures have broadened our understanding of how cells form not only elegant parallel arrays but also more complex MT configurations, including the prominent MT bundles found in preprophase bands, leaf epidermal cells, and developing xylem.     

Severing at sites of microtubule crossover contributes to microtubule alignment in cortical arrays

The cortical microtubule (MT) array and its organization is important in defining the growth axes of plant cells. In roots, the MT array exhibits a net-like configuration in the division zone, and a densely-packed transverse alignment in the elongation zone. This transition is essential for anisotropic cell expansion and consequently has been the subject of intense study. Cotyledons exhibit a net-like array in pavement cells and a predominantly aligned array in the petioles, and provide an excellent system for determining the basis of plant MT organization. We show that in both kinds of MT array, growing MTs frequently encounter existing MTs. Although some steep-angled encounters result in catastrophes, the most frequent outcome of these encounters is successful negotiation of the existing MT by the growing MT to form an MT crossover. Surprisingly, the outcome of such encounters is similar in both aligned and net-like arrays. In contrast, aligned arrays exhibit a much higher frequency of MT severing events compared with net-like arrays. Severing events occur almost exclusively at sites where MTs cross over one another. This process of severing at sites of MT crossover results in the removal of unaligned MTs, and is likely to form the basis for the difference between a net-like and an aligned MT array.     

Microtubule and katanin-dependent dynamics of microtubule nucleation complexes in the acentrosomal Arabidopsis cortical array

Microtubule nucleation in interphase plant cells primarily occurs through branching from pre-existing microtubules at dispersed sites in the cell cortex. The minus ends of new microtubules are often released from the sites of nucleation, and the free microtubules are then transported to new locations by polymer treadmilling. These nucleation-and-release events are characteristic features of plant arrays in interphase cells, but little is known about the spatiotemporal control of these events by nucleating protein complexes. We visualized the dynamics of two fluorescently-tagged γ-tubulin complex proteins, GCP2 and GCP3, in Arabidopsis thaliana. These probes labelled motile complexes in the cytosol that transiently stabilized at fixed locations in the cell cortex. Recruitment of labelled complexes occurred preferentially along existing cortical microtubules, from which new microtubule was synthesized in a branching manner, or in parallel to the existing microtubule. Complexes localized to microtubules were approximately 10-fold more likely to display nucleation than were complexes recruited to other locations. Nucleating complexes remained stable until daughter microtubules were either completely depolymerized from their plus ends or released by katanin-dependent severing activity. These observations suggest that the nucleation complexes are primarily activated on association with microtubule lattices, and that nucleation complex stability depends on association with daughter microtubules and is regulated in part by katanin activity.     

Efficient formation of bipolar microtubule bundles requires microtubule-bound gamma-tubulin complexes

The mechanism for forming linear microtubule (MT) arrays in cells such as neurons, polarized epithelial cells, and myotubes is not well understood. A simpler bipolar linear array is the fission yeast interphase MT bundle, which in its basic form contains two MTs that are bundled at their minus ends. Here, we characterize mto2p as a novel fission yeast protein required for MT nucleation from noncentrosomal gamma-tubulin complexes (gamma-TuCs). In interphase mto2Delta cells, MT nucleation was strongly inhibited, and MT bundling occurred infrequently and only when two MTs met by chance in the cytoplasm. In wild-type 2, we observed MT nucleation from gamma-TuCs bound along the length of existing MTs. We propose a model on how these nucleation events can more efficiently drive the formation of bipolar MT bundles in interphase. Key to the model is our observation of selective antiparallel binding of MTs, which can both explain the generation and spatial separation of multiple bipolar bundles.     

Effects of {gamma}-tubulin complex proteins on microtubule nucleation and catastrophe in fission yeast

Although gamma-tubulin complexes (gamma-TuCs) are known as microtubule (MT) nucleators, their function in vivo is still poorly defined. Mto1p (also known as mbo1p or mod20p) is a gamma-TuC-associated protein that recruits gamma-TuCs specifically to cytoplasmic MT organizing centers (MTOCs) and interphase MTs. Here, we investigated gamma-TuC function by analyzing MT behavior in mto1Delta and alp4 (GCP2 homologue) mutants. These cells have free, extra-long interphase MTs that exhibit abnormal behaviors such as cycles of growth and breakage, MT sliding, treadmilling, and hyperstability. The plus ends of interphase and spindle MTs grow continuously, exhibiting catastrophe defects that are dependent on the CLIP170 tip1p. The minus ends of interphase MTs exhibit shrinkage and pauses. As mto1Delta mutants lack cytoplasmic MTOCs, cytoplasmic MTs arise from spindle or other intranuclear MTs that exit the nucleus. Our findings show that mto1p and gamma-TuCs affect multiple properties of MTs including nucleation, nuclear attachment, plus-end catastrophe, and minus-end shrinkage.     

Development of the preprophase band from random cytoplasmic microtubules in guard mother cells of Allium cepa L.

The organization of microtubule (MT) arrays in the guard mother cells (GMCs) of A. cepa was examined, focussing on the stage at which a longitudinal preprophase band (PPB) is established perpendicular to all other division planes in the epidermis. In the majority of young GMCs, including those seen just after asymmetric division, MTs are distributed randomly throughout the cortex and inner regions of the cytoplasm. Few MTs are associated with the nuclear surface. As the GMCs continue to develop, MTs cluster around the nucleus and a PPB appears as a wide longitudinal band. Microtubules also become prominent between the nucleus and the periclinal and transverse walls, while they decrease in number along the radial longitudinal walls. The PPB progressively narrows by early prophase, and a transversely oriented spindle gradually ensheaths the nucleus. These observations indicate that the initial, broad PPB is organized by a rearrangement of the random cytoplasmic array of MTs. Additional reorganization is responsible for MTs linking the nucleus and the cortex in the future plane of the cell plate, and for narrowing of the PPB.Abbreviations GMC guard mother cell - MT microtubule - PPB preprophase band     

CLASP1 and CLASP2 bind to EB1 and regulate microtubule plus-end dynamics at the cell cortex

CLIP-associating protein (CLASP) 1 and CLASP2 are mammalian microtubule (MT) plus-end binding proteins, which associate with CLIP-170 and CLIP-115. Using RNA interference in HeLa cells, we show that the two CLASPs play redundant roles in regulating the density, length distribution and stability of interphase MTs. In HeLa cells, both CLASPs concentrate on the distal MT ends in a narrow region at the cell margin. CLASPs stabilize MTs by promoting pauses and restricting MT growth and shortening episodes to this peripheral cell region. We demonstrate that the middle part of CLASPs binds directly to EB1 and to MTs. Furthermore, we show that the association of CLASP2 with the cell cortex is MT independent and relies on its COOH-terminal domain. Both EB1- and cortex-binding domains of CLASP are required to promote MT stability. We propose that CLASPs can mediate interactions between MT plus ends and the cell cortex and act as local rescue factors, possibly through forming a complex with EB1 at MT tips.     

Microtubule Cytoskeleton Remodeling by Acentriolar Microtubule-organizing Centers at the Entry and Exit from Mitosis in Drosophila Somatic Cells

Cytoskeleton microtubules undergo a reversible metamorphosis as cells enter and exit mitosis to build a transient mitotic spindle required for chromosome segregation. Centrosomes play a dominant but dispensable role in microtubule (MT) organization throughout the animal cell cycle, supporting the existence of concurrent mechanisms that remain unclear. Here we investigated MT organization at the entry and exit from mitosis, after perturbation of centriole function in Drosophila S2 cells. We found that several MTs originate from acentriolar microtubule-organizing centers (aMTOCs) that contain γ-tubulin and require Centrosomin (Cnn) for normal architecture and function. During spindle assembly, aMTOCs associated with peripheral MTs are recruited to acentriolar spindle poles by an Ncd/dynein-dependent clustering mechanism to form rudimentary aster-like structures. At anaphase onset, down-regulation of CDK1 triggers massive formation of cytoplasmic MTs de novo, many of which nucleated directly from aMTOCs. CDK1 down-regulation at anaphase coordinates the activity of Msps/XMAP215 and the kinesin-13 KLP10A to favor net MT growth and stability from aMTOCs. Finally, we show that microtubule nucleation from aMTOCs also occurs in cells containing centrosomes. Our data reveal a new form of cell cycle–regulated MTOCs that contribute for MT cytoskeleton remodeling during mitotic spindle assembly/disassembly in animal somatic cells, independently of centrioles.     

A role for oxysterol-binding protein-related protein 5 in endosomal cholesterol trafficking

Cytoskeletal organization is central to establishing cell polarity in various cellular contexts, including during messenger ribonucleic acid sorting in Drosophila melanogaster oocytes by microtubule (MT)-dependent molecular motors. However, MT organization and dynamics remain controversial in the oocyte. In this paper, we use rapid multichannel live-cell imaging with novel image analysis, tracking, and visualization tools to characterize MT polarity and dynamics while imaging posterior cargo transport. We found that all MTs in the oocyte were highly dynamic and were organized with a biased random polarity that increased toward the posterior. This organization originated through MT nucleation at the oocyte nucleus and cortex, except at the posterior end of the oocyte, where PAR-1 suppressed nucleation. Our findings explain the biased random posterior cargo movements in the oocyte that establish the germline and posterior.     

Microtubule treadmilling: what goes around comes around

“I'll see it when I believe it” Daniel Mazia Microtubules are centrally involved in many essential cell functions, including mitosis, vesicle motility, and the control of morphogenesis. Further, they appear to be involved in the control of cell cycle progression. To carry out these tasks properly, microtubules assume a protean array of different stability states and degrees of organization and they respond rapidly to requirements of the cell by modification of their organization and stability. In the typical fibroblast cell in culture, microtubules rapidly exchange their subunits with tubulin in the cytoplasmic pool, and control of this rapid turnover appears to be essential to their intrinsic capacity to perform such tasks as the separation of chromosomes in mitosis. Microtubules are not simple equilibrium polymers, but rather, they are capable of unusual nonequilibrium dynamic behaviors. One such behavior, termed treadmilling, involving the intrinsic flow of subunits from one polymer end to the other, is created by differences in the critical subunit concentrations at the opposite microtubule ends. Treadmilling was considered by many to be an in vitro dynamic behavior that did not play an important role in microtubule function in cells. However, recent evidence has established that treadmilling is a major in vivo mechanism underlying the dynamics of microtubule arrays. BioEssays 20 :830–836, 1998. © 1998 John Wiley & Sons, Inc.     

Cytoskeletal changes during resumption of tip growth in nongrowing protonemal cells of the fernAdiantum capillus-veneris L.

Summary Nongrowing, two-celled protonemata of the fernAdiantum capillus-veneris L. resume tip growth within the apical cell upon irradiation with red light. In this study, the phenomenon of growth resumption was analyzed with reference to changes in cytoskeletal organization. Continuous observations of apical cells with time lapse video-microscopy revealed that the nucleus migrated toward the tip ca. 1.9 h after the onset of red light, much earlier than the initiation of tip growth, which took place ca. 8.5 h after irradiation. Cytoskeletal organization was observed at various time points during growth resumption by fluorescent staining of microfilaments (MFs) and microtubules (MTs) with rhodamine-phalloidin and anti-tubulin antibodies. At 2 h after red-light irradiation, endoplasmic MF and MT strands appeared at the apical end of nucleus. These strands extended into the apical endoplasm, where filaments were rare prior to irradiation. Many fine filaments branched from the strands to the cell periphery, including the cortex of the apical-dome region. At this time, cortical circular arrays of MTs and MFs, normally found in the growing apex of protonemal cells, were absent. Both MT and MF circular arrays appeared during the resumption of tip growth concomitantly. The half-maximum appearance of MT and MF circular arrays within a population occurred at 5.4 h and 5.8 h after red-light irradiation, respectively. Thus, the process of red-light-induced resumption of tip growth in fern protonemal cell is composed of a series of events. These events include: (1) the appearance of strands extending from the nucleus toward the apical cortex and the migration of nucleus toward the apex; (2) the formation of circular MT and MF arrays at the sub-apical cortex; and (3) the initiation of cell growth at the apex. These results reflect the significant roles of MF and MT cytoskeleton in the resumption of tip growth.Abbreviations MBS m-maleimidobenzoic acid N-hydroxysuccinimide ester - MF microfilament - MT microtubule     

Stabilization of overlapping microtubules by fission yeast CLASP

Many microtubule (MT) structures contain dynamic MTs that are bundled and stabilized in overlapping arrays. CLASPs are conserved MT-binding proteins implicated in the regulation of MT plus ends. Here, we show that the Schizosaccharomyces pombe CLASP, cls1p/peg1p, mediates the stabilization of overlapping MTs within the mitotic spindle and interphase bundles. cls1p localizes to these regions but not to interphase MT plus ends. Inactivation of cls1p leads to the rapid depolymerization of spindle midzone MTs. cls1p also stabilizes a subset of MTs within interphase bundles. cls1p prevents disassembly of the entire microtubule, while still allowing for plus-end growth. It has no measurable effects on MT nucleation, polymerization, catastrophe, or bundling. A direct interaction with ase1p (PRC1/MAP65) targets cls1p to regions of antiparallel MT overlap. These findings show how a MT-stabilizing factor attached to specific sites on MTs can help to generate MT structures that have both dynamic and stable components.     

Cadherin-mediated regulation of microtubule dynamics

Epithelial polarization and neuronal outgrowth require the assembly of microtubule arrays that are not associated with centrosomes. As these processes generally involve contact interactions mediated by cadherins, we investigated the potential role of cadherin signalling in the stabilization of non-centrosomal microtubules. Here we show that expression of cadherins in centrosome-free cytoplasts increases levels of microtubule polymer and changes the behaviour of microtubules from treadmilling to dynamic instability. This effect is not a result of cadherin expression per se but depends on the formation of cell-cell contacts. The effect of cell-cell contacts is mimicked by application of beads coated with stimulatory anti-cadherin antibody and is suppressed by overexpression of the cytoplasmic cadherin tail. We therefore propose that cadherins initiate a signalling pathway that alters microtubule organization by stabilizing microtubule ends.