Microtubule nucleation and release from the neuronal centrosome

We have proposed that microtubules (MTs) destined for axons and dendrites are nucleated at the centrosome within the cell body of the neuron, and are then released for translocation into these neurites (Baas, P. W., and H. C. Joshi. 1992. J. Cell Biol. 119:171-178). In the present study, we have tested the capacity of the neuronal centrosome to act as a generator of MTs for relocation into other regions of the neuron. In cultured sympathetic neurons undergoing active axonal outgrowth, MTs are present throughout the cell body including the region around the centrosome, but very few (< 10) are directly attached to the centrosome. These results indicate either that the neuronal centrosome is relatively inactive with regard to MT nucleation, or that most of the MTs nucleated at the centrosome are rapidly released. Treatment for 6 h with 10 micrograms/ml nocodazole results in the depolymerization of greater than 97% of the MT polymer in the cell body. Within 5 min after removal of the drug, hundreds of MTs have assembled in the region of the centrosome, and most of these MTs are clearly attached to the centrosome. A portion of the MTs are not attached to the centrosome, but are aligned side-by-side with the attached MTs, suggesting that the unattached MTs were released from the centrosome after nucleation. In addition, unattached MTs are present in the cell body at decreasing levels with increasing distance from the centrosome. By 30 min, the MT array of the cell body is indistinguishable from that of controls. The number of MTs attached to the centrosome is once again diminished to fewer than 10, suggesting that the hundreds of MTs nucleated from the centrosome after 5 min were subsequently released and translocated away from the centrosome. These results indicate that the neuronal centrosome is a highly potent MT- nucleating structure, and provide strong indirect evidence that MTs nucleated from the centrosome are released for translocation into other regions of the neuron.     

Microtubule organization requires cell cycle-dependent nucleation at dispersed cytoplasmic sites: polar and perinuclear microtubule organizing centers in the plant pathogen Ustilago maydis

Growth of most eukaryotic cells requires directed transport along microtubules (MTs) that are nucleated at nuclear-associated microtubule organizing centers (MTOCs), such as the centrosome and the fungal spindle pole body (SPB). Herein, we show that the pathogenic fungus Ustilago maydis uses different MT nucleation sites to rearrange MTs during the cell cycle. In vivo observation of green fluorescent protein-MTs and MT plus-ends, tagged by a fluorescent EB1 homologue, provided evidence for antipolar MT orientation and dispersed cytoplasmic MT nucleating centers in unbudded cells. On budding gamma-tubulin containing MTOCs formed at the bud neck, and MTs reorganized with >85% of all minus-ends being focused toward the growth region. Experimentally induced lateral budding resulted in MTs that curved out of the bud, again supporting the notion that polar growth requires polar MT nucleation. Depletion or overexpression of Tub2, the gamma-tubulin from U. maydis, affected MT number in interphase cells. The SPB was inactive in G2 phase but continuously recruited gamma-tubulin until it started to nucleate mitotic MTs. Taken together, our data suggest that MT reorganization in U. maydis depends on cell cycle-specific nucleation at dispersed cytoplasmic sites, at a polar MTOC and the SPB.     

The plus ends of stable microtubules are the exclusive nucleating structures for microtubules in the axon.

Microtubules (MTs) in the axon have a uniform polarity orientation that is recapitulated during recovery from episodes of MT depolymerization (Heidemann, S. R., M. A. Hamborg, S. J. Thomas, B. Song, S. Lindley, and D. Chu. 1984. J. Cell Biol. 99:1289-1295). This tight regulation of their organization indicates that axonal MTs are spatially regulated by discrete nucleating structures comparable in function to the centrosome. Several authors have proposed that an especially stable class of MTs in the axon may serve as these nucleating structures. In a previous report (Baas, P. W., and M. M. Black. 1990. J. Cell Biol. 111:495-509), we determined that the axons of cultured sympathetic neurons contain two classes of MT polymer, stable and labile, that differ in their sensitivity to nocodazole by roughly 35-fold. The stable and labile polymer represent long-lived and recently assembled polymer, respectively. We also determined that these two classes of polymer can be visually distinguished at the immunoelectron microscopic level based on their content of tyrosinated alpha-tubulin: the labile polymer stains densely, while the stable polymer does not stain. In the present study, we have taken advantage of these observations to directly identify MT nucleating structures in the axon. Neuron cultures were treated with nocodazole for 6 h to completely depolymerize the labile polymer in the axon, and substantially shorten the stable polymer. The cultures were then rinsed free of the drug, permitted to reassemble polymer for various periods of time, and prepared for immunoelectron microscopic localization of tyrosinated alpha-tubulin. Serial reconstruction of consecutive thin sections was undertaken to determine the spatial relationship between the stable MTs and the newly assembled polymer. All of the new polymer assembled in direct continuity with the plus ends of stable MTs, indicating that these ends are assembly competent, and hence capable of acting as nucleating structures. Our results further indicate that no self-assembly of MTs occurs in the axon, nor do any MT nucleating structures exist in the axon other than the plus ends of stable MTs. Thus the plus ends of stable MTs are the exclusive nucleating structures for MTs in the axon.     

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.     

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)     

Microtubule reassembly from nucleating fragments during the regrowth of amputated neurites

We have proposed that stable microtubule (MT) fragments that resist depolymerization may serve as nucleating elements for the local control of MT dynamics in the axon (Heidemann, S. R., M. A. Hamborg, S. J. Thomas, B. Song, S. Lindley, and D. Chu, 1984, J. Cell Biol., 99:1289-1295). Here we report evidence that supports this proposal in studies on the role of MTs in the regrowth of neurites from the distal segments of amputated chick sensory neurites. Amputated neurites collapse to "beads" of axoplasm that rapidly regrow (Shaw, G., and D. Bray, 1977, Exp. Cell Res., 104:55-62). We examined both unarrested regrowth and regrowth after MT disassembly by either cold (-5 degrees C for 2 h) or nocodazole (0.1 microgram/ml for 15-20 min). In all these cases regrowth occurred at 3.5-4.5 micron/min with no delay times other than the times to reach 37 degrees C or rinse out the nocodazole. Electron micrographs of untreated beads show many MTs of varying lengths, while those of cold- and nocodazole-treated beads show markedly shorter MTs. The robust regrowth of neurites from beads containing only very short MTs argues against unfurling of intact MTs from the bead into the growing neurite. Electron micrographs of cold-treated beads lysed under conditions that cause substantial MT depolymerization in untreated intact neurites show persistent MT fragments similar to those in unlysed cold-treated beads. We interpret this as evidence that the MT fragments in cold-treated beads are somehow distinct from the majority of the MT mass that had depolymerized. Collapsed neurites treated with a higher dose of nocodazole (1.0 microgram/ml for 15-20 min) were completely devoid of MTs and regrew only after a 15-20 min delay in two cases but never regrew in 11 other cases. We found that MTs did not return in beads treated with 1.0 microgram/ml nocodazole even 30 min after removal of the drug. It was unlikely that the inability of these beads to reassemble MTs was due to incomplete removal of nocodazole in that a much higher dose (20 micrograms/ml nocodazole) could be quickly rinsed from intact neurites. Beads treated with 1.0 microgram/ml nocodazole could, however, be stimulated to reassemble MTs and regrow neurites by treatment with taxol. We conclude that the immediate, robust regrowth of neurites from collapsed beads of axoplasm requires MT nucleation sites to support MT reassembly.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolated Plant Nuclei Nucleate Microtubule Assembly: The Nuclear Surface in Higher Plants Has Centrosome-like Activity

In most eukaryotic cells, microtubules (MTs) are assembled at identified nucleating sites, such as centrosomes or spindle pole bodies. Higher plant cells do not possess such centrosome-like structures. Thus, the fundamental issues of where and how the intracellular plant MTs are nucleated remain highly debatable. A large body of evidence indicates that plant MTs emerge from the nuclear periphery. In this study, we developed an in vitro assay in which isolated maize nuclei nucleate MT assembly at a tubulin concentration (14 [mu]M of neurotubulin) that is not efficient for spontaneous MT assembly. No MT-stabilizing agents, such as taxol or dimethyl sulfoxide, were used. Our model provides evidence that the nuclear surface functions as a MT-nucleating site in higher plant cells. A monoclonal antibody raised against a pericentriolar antigen immunostained the surface of isolated nuclei, and a 100-kD polypeptide in 4 M urea-treated nuclear extracts was detected.     

Three-dimensional structural characterization of centrosomes from early Drosophila embryos

An understanding of the mechanism and structure of microtubule (MT)- nucleating sites within the pericentriolar material (PCM) of the centrosome has been elusive. This is partly due to the difficulty in obtaining large quantities of centrosomes for analysis, as well as to the problem of attaining interpretable structural data with conventional EM techniques. We describe a protocol for isolating a large quantity of functional centrosomes from early Drosophila embryos. Using automated electron tomography, we have begun a three-dimensional structural characterization of these intact centrosomes with and without regrown MTs. Reconstructions of the centrosomes to approximately 6-8 nm resolution revealed no large structures at the minus ends of MTs, suggesting that if MT-nucleating material physically contacts the MTs, it must conform closely to the shape of the minus end. While many MTs originate near the centrioles, MT minus ends were found throughout the PCM, and even close to its outer boundary. The MTs criss-crossed the PCM, suggesting that nucleating sites are oriented in many different directions. Reconstructions of centrosomes without MTs suggest that there is a reorganization of the PCM upon MT regrowth; moreover, ring-like structures that have a similar diameter as MTs are apparent in the PCM of centrosomes without MTs, and may be MT- nucleating sites.     

The kinetically dominant assembly pathway for centrosomal asters in Caenorhabditis elegans is gamma-tubulin dependent

gamma-Tubulin-containing complexes are thought to nucleate and anchor centrosomal microtubules (MTs). Surprisingly, a recent study (Strome, S., J. Powers, M. Dunn, K. Reese, C.J. Malone, J. White, G. Seydoux, and W. Saxton. Mol. Biol. Cell. 12:1751-1764) showed that centrosomal asters form in Caenorhabditis elegans embryos depleted of gamma-tubulin by RNA-mediated interference (RNAi). Here, we investigate the nucleation and organization of centrosomal MT asters in C. elegans embryos severely compromised for gamma-tubulin function. We characterize embryos depleted of approximately 98% centrosomal gamma-tubulin by RNAi, embryos expressing a mutant form of gamma-tubulin, and embryos depleted of a gamma-tubulin-associated protein, CeGrip-1. In all cases, centrosomal asters fail to form during interphase but assemble as embryos enter mitosis. The formation of these mitotic asters does not require ZYG-9, a centrosomal MT-associated protein, or cytoplasmic dynein, a minus end-directed motor that contributes to self-organization of mitotic asters in other organisms. By kinetically monitoring MT regrowth from cold-treated mitotic centrosomes in vivo, we show that centrosomal nucleating activity is severely compromised by gamma-tubulin depletion. Thus, although unknown mechanisms can support partial assembly of mitotic centrosomal asters, gamma-tubulin is the kinetically dominant centrosomal MT nucleator.     

Method of modelling intracellular transport in branching neurites: application to axons and dendrites of Drosophila sensory neurons

This paper develops a method of calculating the transport of intracellular organelles in neurons with branching neurites which is based on the Smith–Simmons equations of motor-assisted transport. The method is aimed at understanding the effects of microtubule (MT) polarity orientation in branching neurites on transport of organelles at the fundamental level. The method is applied to calculating the organelle transport in axons and dendrites of Drosophila neurons, using the map of MT orientation in such neurons developed by Stone et al. (Mol Biol Cell 19:4122–4129, 2008). The proximal dendrite is assumed to branch and form two distal dendrites. Two different MT polarity arrangements in a proximal dendrite are considered, and implications of these MT arrangements on organelle transport are analysed. It is demonstrated that the MT arrangement found in Drosophila dendrites (MTs have their minus ends out in a proximal dendrite) results in much more efficient motor-driven transport than the structure with a mixed MT orientation in proximal dendrites.     

Microtubule organization and distribution of gamma-tubulin in male meiosis of lepidoptera

Meiotic spindles in males of higher Lepidotera are unusual in that the bulk of the spindle microtubules (MTs) ends about halfway between the equatorial plate and the centrosomes in metaphase. It appears worthwhile to determine how the MTs are nucleated, while their pole proximal ends are distant from the centrosomes. To this end, spermatocytes of Phragmatobia fuliginosa (Arctiidae), collected in the field, were double-labeled with antibodies to beta- and gamma-tubulin. The former antibody reveals the entire microtubular cytoskeleton, and the latter is directed against a newly-discovered tublin isoform that is prevalent in microtubule-organizing centers (MTOCs). The immunocytochemical work was supplemented by a fine structural analysis of MTOCs and spindles. Gamma-tubulin was clearly detected at the spindle poles, and prominent microtubular asters originated from these sites. Additionally, MT arrays at both sides of the equatorial plate in metaphase spermatocytes contained gamma-tubulin. The staining persisted in late anaphase, when kinetochore MTs are depolymerized. This indicates that at least nonkinetochore MTs contain gamma-tubulin. The analysis of ultrathin sections through spindles revealed large amounts of pericentriolar material at the spindles poles, in prometaphase through anaphase. The spindle MTs appeared as regular, straight elements in longitudinal sections. We assume that gamma-tubulin is located at the pole proximal ends of the MTs and/or is associated with the spindle MTs throughout their lengths. In order to distinguish between these possibilities, testes of Ephestia kuehniella (Pyralidae), a laboratory species, were cold-treated prior to double-labeling with antibodies to beta- and gamma-tubulin. The treatment was expected to depolymerize MTs. Astral MTs, which were nucleated end-on by gamma-tubulin-containing material, indeed depolymerized. In contrast, the gamma-tubulin-containing spindle MTs persisted. It is, therefore, conceivable that gamma-tubulin is associated with MTs throughout their lengths in male meiosis of Lepidoptera species. It is plausible that this association stabilizes the MTs against cold-induced disassembly. © 1996 Wiley-Liss, Inc.     

Transport of dendritic microtubules establishes their nonuniform polarity orientation

The immature processes that give rise to both axons and dendrites contain microtubules (MTs) that are uniformly oriented with their plus- ends distal to the cell body, and this pattern is preserved in the developing axon. In contrast, developing dendrites gradually acquire nonuniform MT polarity orientation due to the addition of a subpopulation of oppositely oriented MTs (Baas, P. W., M. M. Black, and G. A. Banker. 1989. J. Cell Biol. 109:3085-3094). In theory, these minus-end-distal MTs could be locally nucleated and assembled within the dendrite itself, or could be transported into the dendrite after their nucleation within the cell body. To distinguish between these possibilities, we exposed cultured hippocampal neurons to nanomolar levels of vinblastine after one of the immature processes had developed into the axon but before the others had become dendrites. At these levels, vinblastine acts as a kinetic stabilizer of MTs, inhibiting further assembly while not substantially depolymerizing existing MTs. This treatment did not abolish dendritic differentiation, which occurred in timely fashion over the next two to three days. The resulting dendrites were flatter and shorter than controls, but were identifiable by their ultrastructure, chemical composition, and thickened tapering morphology. The growth of these dendrites was accompanied by a diminution of MTs from the cell body, indicating a net transfer of MTs from one compartment into the other. During this time, minus-end-distal microtubules arose in the experimental dendrites, indicating that new MT assembly is not required for the acquisition of nonuniform MT polarity orientation in the dendrite. Minus-end-distal microtubules predominated in the more proximal region of experimental dendrites, indicating that most of the MTs at this stage of development are transported into the dendrite with their minus-ends leading. These observations indicate that transport of MTs from the cell body is an essential feature of dendritic development, and that this transport establishes the nonuniform polarity orientation of MTs in the dendrite.     

Mechanical Regulation of Neurite Polarization and Growth: A Computational Study

The densely packed microtubule (MT) array found in neuronal cell projections (neurites) serves two fundamental functions simultaneously: it provides a mechanically stable track for molecular motor-based transport and produces forces that drive neurite growth. The local pattern of MT polarity along the neurite shaft has been found to differ between axons and dendrites. In axons, the neurons’ dominating long projections, roughly 90% of the MTs orient with their rapidly growing plus end away from the cell body, whereas in vertebrate dendrites, their orientations are locally mixed. Molecular motors are known to be responsible for cytoskeletal ordering and force generation, but their collective function in the dense MT cytoskeleton of neurites remains elusive. We here hypothesized that both the polarity pattern of MTs along the neurite shaft and the shaft’s global extension are simultaneously driven by molecular motor forces and should thus be regulated by the mechanical load acting on the MT array as a whole. To investigate this, we simulated cylindrical bundles of MTs that are cross-linked and powered by molecular motors by iteratively solving a set of force-balance equations. The bundles were subjected to a fixed load arising from actively generated tension in the actomyosin cortex enveloping the MTs. The magnitude of the load and the level of motor-induced connectivity between the MTs have been varied systematically. With an increasing load and decreasing motor-induced connectivity between MTs, the bundles became wider in cross section and extended more slowly, and the local MT orientational order was reduced. These results reveal two, to our knowledge, novel mechanical factors that may underlie the distinctive development of the MT cytoskeleton in axons and dendrites: the cross-linking level of MTs by motors and the load acting on this cytoskeleton during growth.     

A composite model for establishing the microtubule arrays of the neuron

Neurons generate two distinct types of processes, termed axons and dendrites, both of which rely on a highly organized array of microtubules for their growth and maintenance. Axonal microtubules are uniformly oriented with their plus ends distal to the cell body, whereas dendritic microtubules are nonuniformly oriented. In neither case are the microtubules attached to the centrosome or any detectable structure that could establish their distinct patterns of polarity orientation. Studies from our laboratory over the past few years have led us to propose the following model for the establishment of the axonal and dendritic microtubule arrays. Microtubules destined for these processes are nucleated at the centrosome within the cell body of the neuron and rapidly released. The released microtubules are then transported into developing axons and dendrites to support their growth. Early in neuronal development, the microtubules are transported with their plus ends leading into immature processes that are the common progenitors of both axons and dendrites. This sets up a uniformly plus-end-distal pattern of polarity orientation, which is preserved in the developing axon. In the case of the dendrite, the plus-end-distal microtubules are joined by another population of microtubules that are transported into these processes with their minus-ends leading. Implicit in this model is that neurons have specialized machinery for regulating the release of microtubules from the centrosome and for transporting them with great specificity.     

The cytoskeleton of neurites after microtubule depolymerization

We previously reported a positive correlation between the number of cold-stable microtubules (MTs) remaining after cold treatment of cat sympathetic nerve and the extent to which the original uniform polarity orientation of axonal MTs was recapitulated after rewarming (J cell biol 99 (1984) 1289). We interpreted these data to indicate that cold-stable fragments, part of larger, generally labile MTs, could act as seeds to organize MT assembly in axons. We report here a direct investigation of the form of cold-stable MTs in neurites of PC-12 cells using two-dimensional reconstruction of serial thin sections. Our data provides strong support for our previous interpretation. The number of MTs in cold-treated neurites was 2-3 times as great while the total length of polymer was approximately half that in control neurites. The average length of MTs in cold-treated neurites was 7-10 times lower than in control neurites. We observed that treatments that depolymerize axonal microtubules cause a marked increase in the number of membranous elements within the axoplasm. This may, however, be a non-specific result of an insult to the axon, since such changes have also been observed in severed, regenerating nerve fibres. We observed that neuroblastoma neurites respond to MT-depolymerization stimuli by developing lateral filopodia similar to those observed in chick dorsal root ganglion cells. Ultrastructural observation of detergent-lysed, whole mounted neuroblastoma (Neuro 2A) cells indicated that the cytoskeleton remaining after MT depolymerization splayed out perpendicular to the long axis of the neurite. That is, we were able to observe many more cytoskeletal 'ends' after MT depolymerization. The concomitant production of filopodia and the splaying of the cytoskeleton after MT depolymerization supports the hypothesis put forward by Wessels et al. (Exp cell res 117 (1978) 335) that the presence or absence of cytoskeletal ends regulates which region of the cell surface is involved in motile behaviour.     

Monastrol, a selective inhibitor of the mitotic kinesin Eg5, induces a distinctive growth profile of dendrites and axons in primary cortical neuron cultures

Various factors including some motor proteins regulate microtubule (MT) transport and influence the formation of neuronal processes. Eg5, a slow and non-processive (+)-end directed motor molecule, is expressed in developing and differentiated neurons. However, how Eg5 works in neurons is still elusive. Thus, we treated primary rat cortical neuron cultures with monastrol, a specific inhibitor of Eg5, to investigate its role in neurons. Immature neurons treated with monastrol extended longer processes than control within a few hours. After 3 days, immature neurons treated with monastrol had longer dendrites but slightly shorter axons than control. This difference in growth between dendrites and axons became more prominent as the cells differentiated until 5 days. Interestingly, MT distributions in the cell bodies of monastrol-treated neurons appeared somewhat circular surrounding the nucleus, while MTs in the cell bodies of control neurons were primarily distributed in the MT organizing center (MTOC) just beside the nucleus. In mature neurons, monastrol treatment induced the axonal clusters of tubulins, grossly not affecting dendrites. Taken together, we conclude that Eg5 acts distinctively on dendrites and axons in neurons and suggest a putative model of how Eg5 works distinctively on dendrites and axons.     

The effect of taxol on centrosome function and microtubule organization in apical cells of Sphacelaria rigidula (Phaeophyceae)

Treatment of interphase apical cells of Sphacelaria rigidula Kützing with 10 μmol L^?1 taxol for 4 h induced drastic changes in microtubule (MT) organization. In normal cells these MTs converge on the centrosomes and are nucleated from the pericentriolar area. After treatment, the endoplasmic, perinuclear and centrosome‐associated MT almost disappeared, and a massive assembly of cortical/subcortical, well‐organized MT bundles was observed. The bundles tended to be axially oriented, usually following the cylindrical wall, although other orientations were not excluded. The MTs in the apical part of the cell seemed to reach the cortex of the apical dome, sometimes bending to follow its curvature, whereas those in the basal portion of the cell terminated close to the transverse wall. Mitotic cells were also highly affected. Typical metaphase stages were very rarely found, and typical anaphase arrangements of chromosomes were completely absent. The chromosomes usually appeared to be dispersed singly or in small groups. Different atypical mitotic configurations were observed, depending on the stage of the cell cycle when the treatment started. The position and the orientation of the atypical mitotic spindles was disturbed. The nuclear envelope was completely disintegrated. The separation of the duplicated centrioles, as well as their usual perinuclear position, was also disturbed. Cortical MT bundles similar to those found in interphase cells were not found in the affected mitotic cells. In contrast, numerous MTs, without definite focal points, were found in the pericentriolar areas. Cytokinesis was inhibited by taxol treatment. The perinuclear and centrosome‐associated MTs found in mitotic cells were gradually replaced by a MT system similar to that of interphase cells. When the cytokinetic diaphragm had already been initiated when taxol treatment began, MTs were found on the cytokinetic plane, a phenomenon not observed in normal untreated cells. The results show clearly that: (i) in interphase cells the ability of centrosomes to nucleate MTs is intensely disturbed by taxol; (ii) centrosome dynamics in MT nucleation vary during the cell cycle; and (iii) taxol strongly affects mitosis and cytokinesis. In addition, it seems that the cortical/subcortical cytoplasm of interphase cells assumes the capacity to form numerous MT bundles.     

Modelling transport of layered double hydroxide nanoparticles in axons and dendrites of cortical neurons

This paper develops a model of nanoparticle transport in neurons. It is assumed that nanoparticles are transported inside endocytic vesicles by a combined effect of dynein-driven transport and diffusion. It is further assumed that in axons nanoparticles are internalised only at axon terminals, whereas in dendrites nanoparticles can enter through the entire plasma membrane. This causes differences in transport of nanoparticles in axons and dendrites; these differences are investigated in this paper. Another difference is microtubule (MT) orientation in axons and dendrites; in axons, all MTs have their plus-ends oriented towards the axon terminal; in a proximal region of a dendrite, MTs have mixed orientation, whereas in a distal dendritic region the MT orientation is similar to that in an axon. It is shown that if molecular-motor-driven transport were powered by dynein alone, such MT orientation in a dendrite would result in a region of nanoparticle accumulation located at the border between the proximal and distal dendritic regions.     

Drosophila CLIP-190 and mammalian CLIP-170 display reduced microtubule plus end association in the nervous system

Axons act like cables, electrically wiring the nervous system. Polar bundles of microtubules (MTs) form their backbones and drive their growth. Plus end–tracking proteins (+TIPs) regulate MT growth dynamics and directionality at their plus ends. However, current knowledge about +TIP functions, mostly derived from work in vitro and in nonneuronal cells, may not necessarily apply to the very different context of axonal MTs. For example, the CLIP family of +TIPs are known MT polymerization promoters in nonneuronal cells. However, we show here that neither Drosophila CLIP-190 nor mammalian CLIP-170 is a prominent MT plus end tracker in neurons, which we propose is due to low plus end affinity of the CAP-Gly domain–containing N-terminus and intramolecular inhibition through the C-terminus. Instead, both CLIP-190 and CLIP-170 form F-actin–dependent patches in growth cones, mediated by binding of the coiled-coil domain to myosin-VI. Because our loss-of-function analyses in vivo and in culture failed to reveal axonal roles for CLIP-190, even in double-mutant combinations with four other +TIPs, we propose that CLIP-190 and -170 are not essential axon extension regulators. Our findings demonstrate that +TIP functions known from nonneuronal cells do not necessarily apply to the regulation of the very distinct MT networks in axons.     

Modelling transport of layered double hydroxide nanoparticles in axons and dendrites of cortical neurons

This paper develops a model of nanoparticle transport in neurons. It is assumed that nanoparticles are transported inside endocytic vesicles by a combined effect of dynein-driven transport and diffusion. It is further assumed that in axons nanoparticles are internalised only at axon terminals, whereas in dendrites nanoparticles can enter through the entire plasma membrane. This causes differences in transport of nanoparticles in axons and dendrites; these differences are investigated in this paper. Another difference is microtubule (MT) orientation in axons and dendrites; in axons, all MTs have their plus-ends oriented towards the axon terminal; in a proximal region of a dendrite, MTs have mixed orientation, whereas in a distal dendritic region the MT orientation is similar to that in an axon. It is shown that if molecular-motor-driven transport were powered by dynein alone, such MT orientation in a dendrite would result in a region of nanoparticle accumulation located at the border between the proximal and distal dendritic regions.