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.     

Dynein-dependent motility of microtubules and nucleation sites supports polarization of the tubulin array in the fungus Ustilago maydis

Microtubules (MTs) are often organized by a nucleus-associated MT organizing center (MTOC). In addition, in neurons and epithelial cells, motor-based transport of assembled MTs determines the polarity of the MT array. Here, we show that MT motility participates in MT organization in the fungus Ustilago maydis. In budding cells, most MTs are nucleated by three to six small and motile gamma-tubulin-containing MTOCs at the boundary of mother and daughter cell, which results in a polarized MT array. In addition, free MTs and MTOCs move rapidly throughout the cytoplasm. Disruption of MTs with benomyl and subsequent washout led to an equal distribution of the MTOC and random formation of highly motile and randomly oriented MTs throughout the cytoplasm. Within 3 min after washout, MTOCs returned to the neck region and the polarized MT array was reestablished. MT motility and polarity of the MT array was lost in dynein mutants, indicating that dynein-based transport of MTs and MTOCs polarizes the MT cytoskeleton. Observation of green fluorescent protein-tagged dynein indicated that this is achieved by off-loading dynein from the plus-ends of motile MTs. We propose that MT organization in U. maydis involves dynein-mediated motility of MTs and nucleation sites.     

Roles for kinesin and myosin during cytokinesis

Cytokinesis in higher plants involves the phragmoplast, a complex cytoplasmic structure that consists of microtubules (MTs), microfilaments (MFs) and membrane elements. Both MTs and MFs are essential for cell plate formation, although it is not clear which motor proteins are involved. Some candidate processes for motor proteins include transport of Golgi vesicles to the plane of the cell plate and the spatiotemporal organization of the cytoskeletal elements in order to achieve proper deposition and alignment of the cell plate. We have focused on the kinesin-like calmodulin binding protein (KCBP) and, more broadly, on myosins. Using an antibody that constitutively activates KCBP, we find that this MT motor, which is minus-end directed, contributes to the organization of the spindle and phragmoplast MTs. It does not participate in vesicle transport; rather, because of the orientation of the phragmoplast MTs, it is supposed that plus-end kinesins fill this role. Myosins, on the other hand, based on their inhibition with 2,3-butanedione monoxime and 1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepine (ML-7), are associated with the process of post-mitotic spindle/phragmoplast alignment and with late lateral expansion of the cell plate. They are also not the principal motors involved in vesicle transport.     

Rho Guanosine Triphosphatase Mediates the Selective Stabilization of Microtubules Induced by Lysophosphatidic Acid

The asymmetric distribution of stable, posttranslationally modified microtubules (MTs) contributes to the polarization of many cell types, yet the factors controlling the formation of these MTs are not known. We have found that lysophosphatidic acid (LPA) is a major serum factor responsible for rapidly generating stable, detyrosinated (Glu) MTs in serum-starved 3T3 cells. Using C3 toxin and val14 rho we showed that rho was both necessary and sufficient for the induction of Glu MTs by LPA and serum. Unlike previously described factors that induce MT stability, rho induced the stabilization of only a subset of the MTs and, in wound-edge cells, these stable MTs were appropriately oriented toward the leading edge of the cell. LPA had little effect on individual parameters of MT dynamics, but did induce long states of pause in a subset of MTs near the edge of the cell. Rho stimulation of MT stability was independent of actin stress fiber formation. These results identify rho as a novel regulator of the MT cytoskeleton that selectively stabilizes MTs during cell polarization by acting as a switch between dynamic and stable states of MTs rather than as a modulator of MT assembly and disassembly.     

Role of cytoplasmic dynein in the axonal transport of microtubules and neurofilaments

Recent studies have shown that the transport of microtubules (MTs) and neurofilaments (NFs) within the axon is rapid, infrequent, asynchronous, and bidirectional. Here, we used RNA interference to investigate the role of cytoplasmic dynein in powering these transport events. To reveal transport of MTs and NFs, we expressed EGFP-tagged tubulin or NF proteins in cultured rat sympathetic neurons and performed live-cell imaging of the fluorescent cytoskeletal elements in photobleached regions of the axon. The occurrence of anterograde MT and retrograde NF movements was significantly diminished in neurons that had been depleted of dynein heavy chain, whereas the occurrence of retrograde MT and anterograde NF movements was unaffected. These results support a cargo model for NF transport and a sliding filament model for MT transport.     

Stable and dynamic microtubules coordinately determine and maintain Drosophila bristle shape

Within interphase cells, microtubules (MTs) are organized in a cell-specific manner to support cell shape and function. Here, we report that coordination between stable and dynamic MTs determines and maintains the highly elongated bristle cell shape. By following MT-decorating hooks and by tracking EB1 we identified two MT populations within bristles: a stable MT population polarized with their minus ends distal to the cell body, and a dynamic MT population that exhibits mixed polarity. Manipulating MT dynamics by Klp10A downregulation demonstrates that MTs can initiate new shaft extensions and thus possess the ability to determine growth direction. Actin filament bundling subsequently supports the newly formed shaft extensions. Analysis of ik2 mutant bristles, established by elongation defects in the Drosophila ikkε homolog, led to the observation that stable and dynamic MT orientation and polarized organization are important for proper bristle elongation. Thus, we demonstrate for the first time that coordination between stable and dynamic MT sets that are axially organized yet differently polarized drives cell elongation.     

Disruption of the Golgi apparatus with brefeldin A does not destabilize the associated detyrosinated microtubule network.

Stable subsets of microtubules (MTs) are often enriched in detyrosinated alpha-tubulin. Recently it has been found that the Golgi apparatus is associated with a subset of relatively stable MTs and that detyrosinated MTs colocalize spatially and temporally with the Golgi apparatus in several cell lines. To determine whether the Golgi apparatus actively stabilizes associated MTs and thus allows their time-dependent detyrosination, we have used the drug brefeldin A (BFA) to disrupt the Golgi apparatus and have monitored changes in the Golgi apparatus and MT populations using simultaneous immunofluorescence and fluorescent lectin microscopy. We found that although BFA caused the Golgi apparatus to completely redistribute to the endoplasmic reticulum (ER), the detyrosinated MTs were not disrupted and remained in a juxtanuclear region. By Western blot analysis we found that even after 6 h of continuous exposure of cells to BFA, there was no detectable reduction in the level of detyrosinated alpha-tubulin. Simultaneous treatment with nocodazole and BFA led to a complete disruption of all MTs and normal Golgi structure/organization. Upon removal of nocodazole in the continued presence of BFA, we found that the detyrosinated MTs reformed in a compact juxtanuclear location in the absence of an intact Golgi complex. Finally, we found that the detyrosinated MTs colocalized precisely with a BFA-resistant structure that binds to the lectin, wheat germ agglutinin. We conclude that the juxtanuclear detyrosinated MTs are not actively stabilized by association with BFA-sensitive Golgi membranes. However, another closely associated structure which binds wheat germ agglutinin may serve to stabilize the juxtanuclear MTs. Alternatively, the MT organizing center (MTOC) and/or MT-associated proteins (MAPs) may organize and stabilize the juxtanuclear detyrosinated MTs.     

Microtubule Patterning in the Presence of Stationary Motor Distributions

In this paper, we construct a novel nonlocal transport model that describes the evolution of microtubules (MTs) as they interact with stationary distributions of motor proteins. An advection term accounts for directed MT transport (sliding due to motor protein action), and an integral term accounts for reorientation of MTs due to their interactions with cross-linking motor proteins. Simulations of our model show how MT patterns depend on boundary constraints, as well as model parameters that represent motor speed, cross-linking capability (motor activity), and directionality. In large domains, and using motor parameter values consistent with experimentally-derived values, we find that patterns such as asters, vortices, and bundles are able to persist. In vivo, MTs take on aster patterns during interphase and they form bundles in neurons and polarized epithelial cells. Vortex patterns have not been observed in vivo, however, are found in in vitro experiments. In constrained domains, we find that similar patterns form (asters, bundles, and vortices). However, we also find that when two opposing motors are present, anti-parallel bundles are able to form, resembling the mitotic spindle during cell division. This model demonstrates how MT sliding and MT reorientation are sufficient to produce experimentally observed patterns.     

A computational model predicts Xenopus meiotic spindle organization

The metaphase spindle is a dynamic bipolar structure crucial for proper chromosome segregation, but how microtubules (MTs) are organized within the bipolar architecture remains controversial. To explore MT organization along the pole-to-pole axis, we simulated meiotic spindle assembly in two dimensions using dynamic MTs, a MT cross-linking force, and a kinesin-5-like motor. The bipolar structures that form consist of antiparallel fluxing MTs, but spindle pole formation requires the addition of a NuMA-like minus-end cross-linker and directed transport of MT depolymerization activity toward minus ends. Dynamic instability and minus-end depolymerization generate realistic MT lifetimes and a truncated exponential MT length distribution. Keeping the number of MTs in the simulation constant, we explored the influence of two different MT nucleation pathways on spindle organization. When nucleation occurs throughout the spindle, the simulation quantitatively reproduces features of meiotic spindles assembled in Xenopus egg extracts.     

Drosophila tubulin-binding cofactor B is required for microtubule network formation and for cell polarity

Microtubules (MTs) are essential for cell division, shape, intracellular transport, and polarity. MT stability is regulated by many factors, including MT-associated proteins and proteins controlling the amount of free tubulin heterodimers available for polymerization. Tubulin-binding cofactors are potential key regulators of free tubulin concentration, since they are required for α-β-tubulin dimerization in vitro. In this paper, we show that mutation of the Drosophila tubulin-binding cofactor B (dTBCB) affects the levels of both α- and β-tubulins and dramatically destabilizes the MT network in different fly tissues. However, we find that dTBCB is dispensable for the early MT-dependent steps of oogenesis, including cell division, and that dTBCB is not required for mitosis in several tissues. In striking contrast, the absence of dTBCB during later stages of oogenesis causes major defects in cell polarity. We show that dTBCB is required for the polarized localization of the axis-determining mRNAs within the oocyte and for the apico-basal polarity of the surrounding follicle cells. These results establish a developmental function for the dTBCB gene that is essential for viability and MT-dependent cell polarity, but not cell division.     

Cell Cycle-Dependent Microtubule-Based Dynamic Transport of Cytoplasmic Dynein in Mammalian Cells

Background

Cytoplasmic dynein complex is a large multi-subunit microtubule (MT)-associated molecular motor involved in various cellular functions including organelle positioning, vesicle transport and cell division. However, regulatory mechanism of the cell-cycle dependent distribution of dynein has not fully been understood.

Methodology/Principal Findings

Here we report live-cell imaging of cytoplasmic dynein in HeLa cells, by expressing multifunctional green fluorescent protein (mfGFP)-tagged 74-kDa intermediate chain (IC74). IC74-mfGFP was successfully incorporated into functional dynein complex. In interphase, dynein moved bi-directionally along with MTs, which might carry cargos such as transport vesicles. A substantial fraction of dynein moved toward cell periphery together with EB1, a member of MT plus end-tracking proteins (+TIPs), suggesting +TIPs-mediated transport of dynein. In late-interphase and prophase, dynein was localized at the centrosomes and the radial MT array. In prometaphase and metaphase, dynein was localized at spindle MTs where it frequently moved from spindle poles toward chromosomes or cell cortex. +TIPs may be involved in the transport of spindle dyneins. Possible kinetochore and cortical dyneins were also observed.

Conclusions and Significance

These findings suggest that cytoplasmic dynein is transported to the site of action in preparation for the following cellular events, primarily by the MT-based transport. The MT-based transport may have greater advantage than simple diffusion of soluble dynein in rapid and efficient transport of the limited concentration of the protein.     

HURP permits MTOC sorting for robust meiotic spindle bipolarity, similar to extra centrosome clustering in cancer cells

In contrast to somatic cells, formation of acentriolar meiotic spindles relies on the organization of microtubules (MTs) and MT-organizing centers (MTOCs) into a stable bipolar structure. The underlying mechanisms are still unknown. We show that this process is impaired in hepatoma up-regulated protein (Hurp) knockout mice, which are viable but female sterile, showing defective oocyte divisions. HURP accumulates on interpolar MTs in the vicinity of chromosomes via Kinesin-5 activity. By promoting MT stability in the spindle central domain, HURP allows efficient MTOC sorting into distinct poles, providing bipolarity establishment and maintenance. Our results support a new model for meiotic spindle assembly in which HURP ensures assembly of a central MT array, which serves as a scaffold for the genesis of a robust bipolar structure supporting efficient chromosome congression. Furthermore, HURP is also required for the clustering of extra centrosomes before division, arguing for a shared molecular requirement of MTOC sorting in mammalian meiosis and cancer cell division.     

A mechanism for nuclear positioning in fission yeast based on microtubule pushing

The correct positioning of the nucleus is often important in defining the spatial organization of the cell, for example, in determining the cell division plane. In interphase Schizosaccharomyces pombe cells, the nucleus is positioned in the middle of the cylindrical cell in an active microtubule (MT)-dependent process. Here, we used green fluorescent protein markers to examine the dynamics of MTs, spindle pole body, and the nuclear envelope in living cells. We find that interphase MTs are organized in three to four antiparallel MT bundles arranged along the long axis of the cell, with MT plus ends facing both the cell tips and minus ends near the middle of the cell. The MT bundles are organized from medial MT-organizing centers that may function as nuclear attachment sites. When MTs grow to the cell tips, they exert transient forces produced by plus end MT polymerization that push the nucleus. After an average of 1.5 min of growth at the cell tip, MT plus ends exhibit catastrophe and shrink back to the nuclear region before growing back to the cell tip. Computer modeling suggests that a balance of these pushing MT forces can provide a mechanism to position the nucleus at the middle of the cell.     

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     

Straighten up and fly right: microtubule dynamics and organization of non-centrosomal arrays in higher plants

Live cell imaging and genetic studies are demonstrating that cortical microtubule arrays in plant cells are dynamic structures in which microtubule (MT) bundles play a key role in creating array organization and function. Steps important for creating and organizing these arrays include recruitment of nucleation complexes to the cell cortex and to the lattices of previously established MTs, association of newly created MTs to the cell cortex, release of MTs from sites of nucleation, transport of released MTs by polymer treadmilling, and subsequent interactions between treadmilling MTs. The results of MT interactions include induced catastrophe, severing, and the capture and reorientation of growing polymer ends by bundling interactions. Together, these properties predict a capacity for self-ordering that is likely to play an important role in establishing the parallel organization of the arrays.     

Adenomatous polyposis coli on microtubule plus ends in cell extensions can promote microtubule net growth with or without EB1

In interphase cells, the adenomatous polyposis coli (APC) protein accumulates on a small subset of microtubules (MTs) in cell protrusions, suggesting that APC may regulate the dynamics of these MTs. We comicroinjected a nonperturbing fluorescently labeled monoclonal antibody and labeled tubulin to simultaneously visualize dynamics of endogenous APC and MTs in living cells. MTs decorated with APC spent more time growing and had a decreased catastrophe frequency compared with non-APC-decorated MTs. Endogenous APC associated briefly with shortening MTs. To determine the relationship between APC and its binding partner EB1, we monitored EB1-green fluorescent protein and endogenous APC concomitantly in living cells. Only a small fraction of EB1 colocalized with APC at any one time. APC-deficient cells and EB1 small interfering RNA showed that EB1 and APC localized at MT ends independently. Depletion of EB1 did not change the growth-stabilizing effects of APC on MT plus ends. In addition, APC remained bound to MTs stabilized with low nocodazole, whereas EB1 did not. Thus, we demonstrate that the association of endogenous APC with MT ends correlates directly with their increased growth stability, that this can occur independently of its association with EB1, and that APC and EB1 can associate with MT plus ends by distinct mechanisms.     

Microtubule plus ends, motors, and traffic of Golgi membranes

The intimate link between microtubule (MT) organization and the components of the secretory pathway has suggested that MT-based motility is an essential component of vesicular membrane transport and membrane polarization. The molecular details of these processes are still under investigation; however, a novel class of MT plus end-binding proteins shed new light on transport between the endoplasmic reticulum (ER) and Golgi apparatus. The dynactin complex, an initial member of this family, shares localization and live-cell imaging phenotypes with other plus end-binding proteins such as CLIP-170 and EB1. In addition, dynactin has been shown to mediate the binding of ER-Golgi transport vesicles to MTs through a regulated MT-binding motif in p150(Glued). Whereas the plus end-binding activity of CLIP-170 and EB1 has been linked to the regulation of dynamic instability, the plus end binding of dynactin is implicated in a search-capture mechanism for dynein-dependent cargoes. An examination of dynactin's role in ER-Golgi transport suggests that plus end binding could be a reflection of fundamental membrane transport mechanisms.     

The transport properties of axonal microtubules establish their polarity orientation

It is well established that axonal microtubules (MTs) are uniformly oriented with their plus ends distal to the neuronal cell body (Heidemann, S. R., J. M. Landers, and M. A. Hamborg. 1981. J. Cell Biol. 91:661-665). However, the mechanisms by which these MTs achieve their uniform polarity orientation are unknown. Current models for axon growth differ with regard to the contributions of MT assembly and transport to the organization and elaboration of the axonal MT array. Do the transport properties or assembly properties of axonal MTs determine their polarity orientation? To distinguish between these possibilities, we wished to study the initiation and outgrowth of axons under conditions that would arrest MT assembly while maintaining substantial levels of preexisting polymer in the cell body that could still be transported into the axon. We found that we could accomplish this by culturing rat sympathetic neurons in the presence of nanomolar levels of vinblastine. In concentrations of the drug up to and including 100 nM, the neurons actively extend axons. The vinblastine- axons are shorter than control axons, but clearly contain MTs. To quantify the effects of the drug on MT mass, we compared the levels of polymer throughout the cell bodies and axons of neurons cultured overnight in the presence of 0, 16, and 50 nM vinblastine with the levels of MT polymer in freshly plated neurons before axon outgrowth. Without drug, the total levels of polymer increase by roughly twofold. At 16 nM vinblastine, the levels of polymer are roughly equal to the levels in freshly plated neurons, while at 50 nM, the levels of polymer are reduced by about half this amount. Thus, 16 nM vinblastine acts as a "kinetic stabilizer" of MTs, while 50 nM results in some net MT disassembly. At both drug concentrations, there is a progressive increase in the levels of MT polymer in the axons as they grow, and a corresponding depletion of polymer from the cell body. These results indicate that highly efficient mechanisms exist in the neuron to transport preassembled MTs from the cell body into the axon. These mechanisms are active even at the expense of the cell body, and even under conditions that promote some MT disassembly in the neuron. MT polarity analyses indicate that the MTs within the vinblastine-axons, like those in control axons, are uniformly plus-end-distal.(ABSTRACT TRUNCATED AT 400 WORDS)     

Providing Positional Information with Active Transport on Dynamic Microtubules

Microtubules (MTs) are dynamic protein polymers that change their length by switching between growing and shrinking states in a process termed dynamic instability. It has been suggested that the dynamic properties of MTs are central to the organization of the eukaryotic intracellular space, and that they are involved in the control of cell morphology, but the actual mechanisms are not well understood. Here, we present a theoretical analysis in which we explore the possibility that a system of dynamic MTs and MT end-tracking molecular motors is providing specific positional information inside cells. We compute the MT length distribution for the case of MT-length-dependent switching between growing and shrinking states, and analyze the accumulation of molecular motors at the tips of growing MTs. Using these results, we show that a transport system consisting of dynamic MTs and associated motor proteins can deliver cargo proteins preferentially to specific positions within the cell. Comparing our results with experimental data in the model organism fission yeast, we propose that the suggested mechanisms could play important roles in setting length scales during cellular morphogenesis.     

Both carboxy-terminal tails of alpha- and beta-tubulin are essential, but either one will suffice

Microtubules (MTs) are organized into distinct systems essential for cell shape, movement, intracellular transport, and division. Electron crystallographic analyses provide little information about how MTs produce diverse structures and functions, perhaps because they failed to visualize the last 10 residues of the alpha- and the last 18 of the beta-tubulin C-terminal tails (CTTs), which likely play a role in MT diversity. CTTs define conserved, nonallelic isotypes in mammals, are major sites of posttranslational modifications (PTMs), are binding sites for microtubule-associated proteins (MAPs), and determine MT motor processivity. Using mutagenesis and homologous gene replacement in Tetrahymena thermophila, we analyzed mutations, deletions, tail switches, and tail duplications of alpha- and beta-tubulin CTTs. We demonstrate that a tail is required for the essential function of both alpha- and beta-tubulin. However, the two tails are interchangeable, and cells grow normally with either an alpha or a beta tail on both tubulins. In addition, an alpha gene containing a duplicated alpha C terminus rescues a lethal mutant lacking all known posttranslational modification sites on the beta C terminus but cannot rescue deletion of the beta tail. Thus, tubulin tails have a second essential function that is not associated with posttranslational modification.