Quantitative analysis of microtubule transport in growing nerve processes

In neurons, tubulin is synthesized primarily in the cell body, whereas the molecular machinery for neurite extension and elaboration of microtubule (MT) array is localized to the growth cone region. This unique functional and biochemical compartmentalization of neuronal cells requires transport mechanisms for the delivery of newly synthesized tubulin and other cytoplasmic components from the cell body to the growing axon. According to the polymer transport model, tubulin is transported along the axon as a polymer. Because the majority of axonal MTs are stationary at any given moment, it has been assumed that only a small fraction of MTs translocates along the axon by saltatory movement reminiscent of the fast axonal transport. Such intermittent "stop and go" MT transport has been difficult to detect or to exclude by using direct video microscopy methods. In this study, we measured the translocation of MT plus ends in the axonal shaft by expressing GFP-EB1 in Xenopus embryo neurons in culture. Formal quantitative analysis of MT assembly/disassembly indicated that none of the MTs in the axonal shaft were rapidly transported. Our results suggest that transport of axonal MTs is not required for delivery of newly synthesized tubulin to the growing nerve processes.     

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.     

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.     

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.     

Newly assembled microtubules are concentrated in the proximal and distal regions of growing axons

We have investigated the sites of microtubule (MT) assembly in neurons during axon growth by taking advantage of the relationship between the proportion of tyrosinated alpha-tubulin (tyr-tubulin) in MTs and their age. Specifically, young (newly assembled) MTs contain more tyr-tubulin than older (more long-lived) MTs. To quantify the relative proportion of tyr-tubulin in MTs, cultured rat sympathetic neurons were permeabilized under conditions that stabilize existing MTs and remove unassembled tubulin. The MTs were then double-stained with antibodies to tyr-tubulin (as a measure of the amount of tyr-tubulin in MTs) and to beta-tubulin (as a measure of total MT mass), using immunofluorescence procedures. Cells were imaged with a cooled charge-coupled device camera and the relative proportion of tyr-tubulin in the MTs was quantified by computing the ratio of the tyr-tubulin fluorescence to the beta-tubulin fluorescence using a novel application of digital image processing and analysis techniques. The amount of tyr-tubulin in the MTs was highest in the cell body and at the growth cone; peak ratios in these two regions were approximately 10-fold higher than for the axon shaft. Moving out from the cell body into the axon, the tyr-tubulin content declined over an average distance of 40 microns to reach a constant low value within the axon shaft and then rose again more distally, over an average distance of 110 microns, to reach a peak at the growth cone (average axon length = 358 microns). These observations indicate that newly assembled MTs are concentrated in the proximal and distal regions of growing axons and therefore that the cell body and growth cone are the most active sites of MT assembly dynamics in neurons that are actively extending axons.     

Dynamics of Axonal Microtubules Regulate the Topology of New Membrane Insertion into the Growing Neurites

Nerve growth depends on the delivery of cell body–synthesized material to the growing neuronal processes. The cellular mechanisms that determine the topology of new membrane addition to the axon are not known. Here we describe a technique to visualize the transport and sites of exocytosis of cell body– derived vesicles in growing axons. We found that in Xenopus embryo neurons in culture, cell body–derived vesicles were rapidly transported all the way down to the growth cone region, where they fused with the plasma membrane. Suppression of microtubule (MT) dynamic instability did not interfere with the delivery of new membrane material to the growth cone region; however, the insertion of vesicles into the plasma membrane was dramatically inhibited. Local disassembly of MTs by focal application of nocodazole to the middle axonal segment resulted in the addition of new membrane at the site of drug application. Our results suggest that the local destabilization of axonal MTs is necessary and sufficient for the delivery of membrane material to specific neuronal sites.     

Individual microtubules in the axon consist of domains that differ in both composition and stability

We have explored the composition and stability properties of individual microtubules (MTs) in the axons of cultured sympathetic neurons. Using morphometric means to quantify the MT mass remaining in axons after various times in 2 micrograms/ml nocodazole, we observed that approximately 48% of the MT mass in the axon is labile, depolymerizing with a t1/2 of approximately 5 min, whereas the remaining 52% of the MT mass is stable, depolymerizing with a t1/2 of approximately 240 min. Immunofluorescence analyses show that the labile MTs in the axon are rich in tyrosinated alpha-tubulin, whereas the stable MTs contain little or no tyrosinated alpha-tubulin and are instead rich in posttranslationally detyrosinated and acetylated alpha-tubulin. These results were confirmed quantitatively by immunoelectron microscopic analyses of the distribution of tyrosinated alpha-tubulin among axonal MTs. Individual MT profiles were typically either uniformly labeled for tyrosinated alpha-tubulin all along their length, or were completely unlabeled. Roughly 48% of the MT mass was tyrosinated, approximately 52% was detyrosinated, and approximately 85% of the tyrosinated MTs were depleted within 15 min of nocodazole treatment. Thus, the proportion of MT profiles that were either tyrosinated or detyrosinated corresponded precisely with the proportion of MTs that were either labile or stable respectively. We also observed MT profiles that were densely labeled for tyrosinated alpha-tubulin at one end but completely unlabeled at the other end. In all of these latter cases, the tyrosinated, and therefore labile domain, was situated at the plus end of the MT, whereas the detyrosinated, and therefore stable domain was situated at the minus end of the MT, and in each case there was an abrupt transition between the two domains. Based on the frequency with which these latter MT profiles were observed, we estimate that minimally 40% of the MTs in the axon are composite, consisting of a stable detyrosinated domain in direct continuity with a labile tyrosinated domain. The extreme drug sensitivity of the labile domains suggests that they are very dynamic, turning over rapidly within the axon. The direct continuity between the labile and stable domains indicates that labile MTs assemble directly from stable MTs. We propose that stable MTs act as MT nucleating structures that spatially regulate MT dynamics in the axon.     

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.     

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.     

Gamma-tubulin distribution in the neuron: implications for the origins of neuritic microtubules

Axons and dendrites contain dense microtubule (MT) assays that are not attached to a traditional MT nucleating structure such as the centrosome. Nevertheless, the MTs within these neurites are highly organized with respect to their polarity, and consist of a regular 13-protofilament lattice, the two known characteristics of MTs nucleated at the centrosome. These observations suggest either that axonal and dendritic MTs arise at the centrosome, or that they are nucleated locally, following a redistribution of MT nucleating material from the centrosome during neuronal development. To begin distinguishing between these possibilities, we have determined the distribution of gamma-tubulin within cultured sympathetic neurons. gamma-tubulin, a newly discovered protein which is specifically localized to the pericentriolar region of nonneuronal cells (Zheng, Y., M. K. Jung, and B. R. Oakley. 1991. Cell. 65:817-823; Stearns, T., L. Evans, and M. Kirschner. 1991. Cell. 65:825-836), has been shown to play a critical role in MT nucleation in vivo (Joshi, H. C., M. J. Palacios, L. McNamara, and D. W. Cleveland. 1992. Nature (Lond.). 356:80-83). Because the gamma-tubulin content of individual cells is extremely low, we relied principally on the high degree of resolution and sensitivity afforded by immunoelectron microscopy. Our studies reveal that, like the situation in nonneuronal cells, gamma-tubulin is restricted to the pericentriolar region of the neuron. Furthermore, serial reconstruction analyses indicate that the minus ends of MTs in both axons and dendrites are free of gamma-tubulin immunoreactivity. The absence of gamma-tubulin from the axon was confirmed by immunoblot analyses of pure axonal fractions obtained from explant cultures. The observation that gamma-tubulin is restricted to the pericentriolar region of the neuron provides compelling support for the notion that MTs destined for axons and dendrites are nucleated at the centrosome, and subsequently released for translocation into these neurites.     

Sorting of cargos between axons and dendrites: modelling of differences in cargo transport in these two types of neurites

Explaining how intracellular cargos are sorted between axons and dendrites is important for a mechanistic understanding of what happens in many neurodegenerative disorders. A simple model of cargo sorting relies on differences in microtubule (MT) orientation between axons and dendrites: in mammalian neurons all MTs in axons have their plus ends directed outward while in proximal regions of dendrites the MT polarity is mixed. It can therefore be assumed that cargos that need to be driven into axons associate with kinesin motors while cargos that need to be driven into dendrites associate with dynein motors. This paper develops equations of cargo transport in axons and dendrites based on the above assumptions. Propagation of a pulse of radiolabelled cargos entering an axon and dendrite is simulated. The model equations are solved utilising the Laplace transform method. Differences in cargo transport between axons and dendrites are discussed.     

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.     

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.     

Tubulin transport in neurons

A question of broad importance in cellular neurobiology has been, how is microtubule cytoskeleton of the axon organized? It is of particular interest because of the history of conflicting results concerning the form in which tubulin is transported in the axon. While many studies indicate a stationary nature of axonal microtubules, a recent series of experiments reports that microtubules are recruited into axons of neurons grown in the presence of a microtubule-inhibitor, vinblastine (Baas, P.W., and F.J. Ahmad. 1993.J. Cell Biol. 120:1427-1437: Ahmad F.J., and P.W. Baas. 1995. J. Cell Sci, 108:2761-2769; Sharp, D.J., W. Yu, and P.W. Baas. 1995. J. Cell Biol, 130:93-103; Yu, W., and P.W. Baas. 1995. J. Neurosci. 15:6827-6833.). Since vinblastine stabilizes bulk microtubule-dynamics in vitro, it was concluded that preformed microtubules moved into newly grown axons. By visualizing the polymerization of injected fluorescent tubulin, we show that substantial microtubule polymerization occurs in neurons grown at reported vinblastine concentrations. Vinblastine inhibits, in a concentration-dependent manner, both neurite outgrowth and microtubule assembly. More importantly, the neuron growth conditions of low vinblastine concentration allowed us to visualize the footprints of the tubulin wave as it polymerized and depolymerized during its slow axonal transport. In contrast, depolymerization resistant fluorescent microtubules did not move when injected in neurons. We show that tubulin subunits, not microtubules, are the primary form of tubulin transport in neurons.     

Microtubule transport and assembly during axon growth

There is controversy concerning the mechanisms by which the axonal microtubule (MT) array is elaborated, with some models focusing on MT assembly and other models focusing on MT transport. We have proposed a composite model in which MT assembly and transport are both important (Joshi, H.C., and P.W. Baas. 1993. J. Cell Biol. 121:1191-1196). In the present study, we have taken a novel approach to evaluate the merits of this proposal. Biotinylated tubulin was microinjected into cultured neurons that had already grown short axons. The axons were then permitted to grow longer, after which the cells were prepared for immunoelectron microscopic analyses. We reasoned that any polymer that assembled or turned over subunits after the introduction of the probe should label for biotin, while any polymer that was already assembled but did not turnover should not label. Therefore, the presence in the newly grown region of the axon of any unlabeled MT polymer is indicative of MT transport. In sampled regions, the majority of the polymer was labeled, indicating that MT assembly events are active during axon growth. Varying amounts of unlabeled polymer were also present in the newly grown regions, indicating that MT transport also occurs. Together these findings demonstrate that MT assembly and transport both contribute to the elaboration of the axonal MT array.     

Development of neurons in the abdominal ganglion of Aplysia californica. I. Axosomatic synaptic contacts.

The development of mariculture techniques for the raising of Aplysia californica in the laboratory from fertilized egg to reproductively mature adult permits the study of the developmental program whereby individual identified neurons in the abdominal ganglion acquire their specific adult properties. In this paper, we describe one of the early steps of this developmental program: the outgrowth of axonal processes by neurons of the abdominal ganglion. Axonal outgrowth is correlated with and may be triggered by the transient appearance of morphologically identifiable axosomatic contacts between the as yet undifferentiated cell body of specific neurons and an axon terminal from an incoming nerve fiber from the pleuroabdominal connective. The evidence that transient axosomatic contacts may signal neuronal differentiation is the following: (1) Axosomatic contacts have not been observed in the abdominal ganglion of adult animals, whereas they are commonly observed during the early stages of development. (2) Cells that receive axosomatic contacts are undifferentiated morphologically and do not as yet have axons. By contrast, cells with axons do not have soma contacts. (3) Individual cells that can be identified from animal to animal in the same and succeeding developmental stages receive axosomatic contacts on similar topographic postions of the cell body at one point in development. Axon outgrowth then occurs at the site of contact. Later in development, with further axon extension, these cells no longer have synaptic contacts on the cell body or axon.     

Defective Neurofilament Transport in Mouse Models of Amyotrophic Lateral Sclerosis: A Review

Neurofilament proteins synthesized in the cell body of neurons are assembled and transported into axons, where they influence axon radial growth, axonal transport, and nerve conduction velocities. In diseased states, neurofilaments accumulate in cell bodies and proximal axons of affected neurons, and these lesions are characteristic of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), spinal muscular atrophy (SMA), Charcot-Marie-Tooth disease type 2 (CMT2), and hereditary sensory motor neuropathy. Although the molecular mechanisms that contribute to these accumulations are not yet identified, transgenic mouse models are beginning to provide insight into the role of neurofilament transport in disease-related dysfunction of neurons. This review addresses axonal transport in mouse models of ALS and the special significance of neurofilament transport in this disease.     

Septins guide noncentrosomal microtubules to promote focal adhesion disassembly in migrating cells

Endothelial cell migration is critical for vascular angiogenesis and is compromised to facilitate tumor metastasis. The migratory process requires the coordinated assembly and disassembly of focal adhesions (FA), actin, and microtubules (MT). MT dynamics at FAs deliver vesicular cargoes and enhance actomyosin contractility to promote FA turnover and facilitate cell advance. Noncentrosomal (NC) MTs regulate FA dynamics and are sufficient to drive cell polarity, but how NC MTs target FAs to control FA turnover is not understood. Here, we show that Rac1 induces the assembly of FA-proximal septin filaments that promote NC MT growth into FAs and inhibit mitotic centromere-associated kinesin (MCAK)-associated MT disassembly, thereby maintaining intact MT plus ends proximal to FAs. Septin-associated MT rescue is coupled with accumulation of Aurora-A kinase and cytoplasmic linker-associated protein (CLASP) localization to the MT between septin and FAs. In this way, NC MTs are strategically positioned to undergo MCAK- and CLASP-regulated bouts of assembly and disassembly into FAs, thereby regulating FA turnover and cell migration.     

Characterization of loss-of-function and gain-of-function Eph receptor tyrosine kinase signaling in C. elegans axon targeting and cell migration

To understand how our brains function, it is necessary to know how neurons position themselves and target their axons and dendrites to their correct locations. Several evolutionarily conserved axon guidance molecules have been shown to help navigate axons to their correct target site. The Caenorhabditis elegans Eph receptor tyrosine kinase (RTK), VAB-1, has roles in early neuroblast and epidermal cell movements, but its roles in axon guidance are not well understood. Here, we report that mutations that disrupt the VAB-1 Eph receptor tyrosine kinase cause incompletely penetrant defects in axonal targeting and neuronal cell body positioning. The predominant axonal defect in vab-1 mutant animals was an overextension axon phenotype. Interestingly, constitutively active VAB-1 tyrosine kinase signaling caused a lack of axon outgrowth or an early termination phenotype, opposite to the loss-of-function phenotype. The combination of loss-of-function and gain-of-function analyses suggests that the VAB-1 Eph RTK is required for targeting or limiting axons and neuronal cells to specific regions, perhaps by transducing a repellent or stop cue.