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1.
Axonal cytoskeletal and cytosolic proteins are synthesized in the neuronal cell body and transported along axons by slow axonal transport, but attempts to observe this movement directly in living cells have yielded conflicting results. Here we report the direct observation of the axonal transport of neurofilament protein tagged with green fluorescent protein in cultured nerve cells. Live-cell imaging of naturally occurring gaps in the axonal neurofilament array reveals rapid, intermittent and highly asynchronous movement of fluorescent neurofilaments. The movement is bidirectional, but predominantly anterograde. Our data indicate that the slow rate of slow axonal transport may be the result of rapid movements interrupted by prolonged pauses. 相似文献
2.
Studies on the axonal transport of neurofilament proteins in cultured neurons have shown they move at fast rates, but their overall rate of movement is slow because they spend most of their time not moving. Using correlative light and electron microscopy, we have shown that these proteins move in the form of assembled neurofilament polymers. However, the polypeptide composition of these moving polymers is not known. To address this, we visualized neurofilaments in cultured neonatal mouse sympathetic neurons using GFP-tagged neurofilament protein M and performed time-lapse fluorescence microscopy of naturally occurring gaps in the axonal neurofilament array. When neurofilaments entered the gaps, we stopped them in their tracks using a rapid perfusion and permeabilization technique and then processed them for immunofluorescence microscopy. To compare moving neurofilaments to the total neurofilament population, most of which are stationary at any point in time, we also performed immunofluorescence microscopy on neurofilaments in detergent-splayed axonal cytoskeletons. All neurofilaments, both moving and stationary, contained NFL, NFM, peripherin and alpha-internexin along>85% of their length. NFH was absent due to low expression levels in these neonatal neurons. These data indicate that peripherin and alpha-internexin are integral and abundant components of neurofilament polymers in these neurons and that both moving and stationary neurofilaments in these neurons are complex heteropolymers of at least four different neuronal intermediate filament proteins. 相似文献
3.
According to the "stop-and-go" hypothesis of slow axonal transport, cytoskeletal and cytosolic proteins are transported along axons at fast rates but the average velocity is slow because the movements are infrequent and bidirectional. To test whether this hypothesis can explain the kinetics of slow axonal transport in vivo, we have developed a stochastic model of neurofilament transport in axons. We propose that neurofilaments move in both anterograde and retrograde directions along cytoskeletal tracks, alternating between short bouts of rapid movement and short "on-track" pauses, and that they can also temporarily disengage from these tracks, resulting in more prolonged "off-track" pauses. We derive the kinetic parameters of the model from a detailed analysis of the moving and pausing behavior of single neurofilaments in axons of cultured neurons. We show that the model can match the shape, velocity, and spreading of the neurofilament transport waves obtained by radioisotopic pulse labeling in vivo. The model predicts that axonal neurofilaments spend approximately 8% of their time on track and approximately 97% of their time pausing during their journey along the axon. 相似文献
4.
Rapid movement of microtubules in axons 总被引:1,自引:0,他引:1
Cytoskeletal and cytosolic proteins are transported along axons in the slow components of axonal transport at average rates of about 0.002-0.1 microm/s. This movement is essential for axonal growth and survival, yet the mechanism is poorly understood. Many studies on slow axonal transport have focused on tubulin, the subunit protein of microtubules, but attempts to observe the movement of this protein in cultured nerve cells have been largely unsuccessful. Here, we report direct observations of the movement of microtubules in cultured nerve cells using a modified fluorescence photobleaching strategy combined with difference imaging. The movements are rapid, with average rates of 1 microm/s, but they are also infrequent and highly asynchronous. These observations indicate that microtubules are propelled along axons by fast motors. We propose that the overall rate of movement is slow because the microtubules spend only a small proportion of their time moving. The rapid, infrequent, and highly asynchronous nature of the movement may explain why the axonal transport of tubulin has eluded detection in so many other studies. 相似文献
5.
We have investigated the movement of green fluorescent protein-tagged neurofilaments at the distal ends of growing axons by using time-lapse fluorescence imaging. The filaments moved in a rapid, infrequent, and asynchronous manner in either an anterograde or retrograde direction (60% anterograde, 40% retrograde). Most of the anterograde filaments entered the growth cone and most of the retrograde filaments originated in the growth cone. In a small number of cases we were able to observe neurofilaments reverse direction, and all of these reversals occurred in or close to the growth cone. We conclude that neurofilament polymers are delivered rapidly and infrequently to the tips of growing axons and that some of these polymers reverse direction in the growth cone and move back into the axon. We propose that 1) growth cones are a preferential site of neurofilament reversal in distal axons, 2) most retrograde neurofilaments in distal axons originate by reversal of anterograde filaments in the growth cone, 3) those anterograde filaments that do not reverse direction are recruited to form the neurofilament cytoskeleton of the newly forming axon, and 4) the net delivery of neurofilament polymers to growth cones may be controlled by regulating the reversal frequency. 相似文献
6.
The classic view of slow axonal transport maintains that microtubules, neurofilaments, and actin filaments move down the axon relatively coherently at rates significantly slower than those characteristic of known motor proteins. Recent studies indicate that the movement of these cytoskeletal polymers is actually rapid, asynchronous, intermittent, and most probably fueled by familiar motors such as kinesins, myosins, and cytoplasmic dynein. This new view, which is supported by both live-cell imaging and mechanistic analyses, suggests that slow axonal transport is both rapid and plastic, and hence could underlie transformations in neuronal morphology. 相似文献
7.
Brown A 《Nature reviews. Molecular cell biology》2000,1(2):153-156
Efforts to observe the slow axonal transport of cytoskeletal polymers during the past decade have yielded conflicting results, and this has generated considerable controversy. The movement of neurofilaments has now been seen, and it is rapid, infrequent and highly asynchronous. This motile behaviour could explain why slow axonal transport has eluded observation for so long. 相似文献
8.
Adam J. Fournier James D. Hogan Labchan Rajbhandari Shiva Shrestha Arun Venkatesan K. T. Ramesh 《PloS one》2015,10(6)
Although a number of cytoskeletal derangements have been described in the setting of traumatic axonal injury (TAI), little is known of early structural changes that may serve to initiate a cascade of further axonal degeneration. Recent work by the authors has examined conformational changes in cytoskeletal constituents of neuronal axons undergoing traumatic axonal injury (TAI) following focal compression through confocal imaging data taken in vitro and in situ. The present study uses electron microscopy to understand and quantify in vitro alterations in the ultrastructural composition of microtubules and neurofilaments within neuronal axons of rats following focal compression. Standard transmission electron microscopy processing methods are used to identify microtubules, while neurofilament identification is performed using antibody labeling through gold nanoparticles. The number, density, and spacing of microtubules and neurofilaments are quantified for specimens in sham Control and Crushed groups with fixation at <1min following load. Our results indicate that the axon caliber dependency known to exist for microtubule and neurofilament metrics extends to axons undergoing TAI, with the exception of neurofilament spacing, which appears to remain constant across all Crushed axon diameters. Confidence interval comparisons between Control and Crushed cytoskeletal measures suggests early changes in the neurofilament spatial distributions within axons undergoing TAI may precede microtubule changes in response to applied loads. This may serve as a trigger for further secondary damage to the axon, representing a key insight into the temporal aspects of cytoskeletal degeneration at the component level, and suggests the rapid removal of neurofilament sidearms as one possible mechanism. 相似文献
9.
The organization of the axonal cytoskeleton is a key determinant of the normal function of an axon, which is a long thin projection of a neuron. Under normal conditions two axonal cytoskeletal polymers, microtubules and neurofilaments, align longitudinally in axons and are interspersed in axonal cross-sections. However, in many neurotoxic and neurodegenerative disorders, microtubules and neurofilaments segregate apart from each other, with microtubules and membranous organelles clustered centrally and neurofilaments displaced to the periphery. This striking segregation precedes the abnormal and excessive neurofilament accumulation in these diseases, which in turn leads to focal axonal swellings. While neurofilament accumulation suggests an impairment of neurofilament transport along axons, the underlying mechanism of their segregation from microtubules remains poorly understood for over 30 years. To address this question, we developed a stochastic multiscale model for the cross-sectional distribution of microtubules and neurofilaments in axons. The model describes microtubules, neurofilaments and organelles as interacting particles in a 2D cross-section, and is built upon molecular processes that occur on a time scale of seconds or shorter. It incorporates the longitudinal transport of neurofilaments and organelles through this domain by allowing stochastic arrival and departure of these cargoes, and integrates the dynamic interactions of these cargoes with microtubules mediated by molecular motors. Simulations of the model demonstrate that organelles can pull nearby microtubules together, and in the absence of neurofilament transport, this mechanism gradually segregates microtubules from neurofilaments on a time scale of hours, similar to that observed in toxic neuropathies. This suggests that the microtubule-neurofilament segregation can be a consequence of the selective impairment of neurofilament transport. The model generates the experimentally testable prediction that the rate and extent of segregation will be dependent on the sizes of the moving organelles as well as the density of their traffic. 相似文献
10.
Control of axonal caliber by neurofilament transport 总被引:30,自引:13,他引:17
The role of neurofilaments, the intermediate filaments of nerve cells, has been conjectural. Previous morphological studies have suggested a close relationship between neurofilament content and axonal caliber. In this study, the regenerating neuron was used as a model system for testing the hypotheses that neurofilaments are intrinsic determinants of axonal caliber, and that neurofilament content is controlled by the axonal transport of neurofilaments. This system was chosen because previous studies had shown that, after axotomy, axonal caliber was reduced within the proximal stump of the regenerating nerve and, because the relative amount of neurofilament protein undergoing axonal transport in regenerating axons was selectively reduced. The relationship between axonal caliber and neurofilament number was examined in a systematic fashion in both regenerating and control motor axons in rat L5 ventral root. Reconstruction of the spatial and temporal sequences of axonal atrophy in the proximal stump after axotomy showed that reductions in axonal caliber were first detected in the most proximal region of the root and subsequently progressed in a proximal-to-distal direction at a rate of 1.7 mm/day, which is identical to the rate of neurofilament transport in these neurons. Quantitative ultrastructural studies showed that these reductions in caliber correlated with a proportional decrease in the number of axonal neurofilaments but not microtubules. These results support the hypotheses that neurofilament content is a major intrinsic determinant of axonal caliber and that neurofilament content is controlled by the axonal transport of neurofilaments. On this basis, we suggest a role for neurofilaments in the control of axonal volume. 相似文献
11.
Tight Functional Coupling of Kinesin-1A and Dynein Motors in the Bidirectional Transport of Neurofilaments 下载免费PDF全文
We have tested the hypothesis that kinesin-1A (formerly KIF5A) is an anterograde motor for axonal neurofilaments. In cultured sympathetic neurons from kinesin-1A knockout mice, we observed a 75% reduction in the frequency of both anterograde and retrograde neurofilament movement. This transport defect could be rescued by kinesin-1A, and with successively decreasing efficacy by kinesin-1B and kinesin-1C. In wild-type neurons, headless mutants of kinesin-1A and kinesin-1C inhibited both anterograde and retrograde movement in a dominant-negative manner. Because dynein is thought to be the retrograde motor for axonal neurofilaments, we investigated the effect of dynein inhibition on anterograde and retrograde neurofilament transport. Disruption of dynein function by using RNA interference, dominant-negative approaches, or a function-blocking antibody also inhibited both anterograde and retrograde neurofilament movement. These data suggest that kinesin-1A is the principal but not exclusive anterograde motor for neurofilaments in these neurons, that there may be some functional redundancy among the kinesin-1 isoforms with respect to neurofilament transport, and that the activities of the anterograde and retrograde neurofilament motors are tightly coordinated. 相似文献
12.
Local modulation of neurofilament phosphorylation, axonal caliber, and slow axonal transport by myelinating Schwann cells. 总被引:27,自引:0,他引:27
Studies in Trembler and control mice demonstrated that myelinating Schwann cells exert a profound influence on axons. Extensive contacts between myelin and axons have been considered structural. However, demyelination decreases neurofilament phosphorylation, slow axonal transport, and axonal diameter, as well as significantly increasing neurofilament density. In control sciatic nerves with grafted Trembler nerve segments, these changes were spatially restricted: they were confined to axon segments without normal myelination. Adjacent regions of the same axons had normal diameters, neurofilament phosphorylation, cytoskeletal organization, and axonal transport rates. Close intercellular contacts between myelinating Schwann cells and axons modulate a kinase-phosphatase system acting on neurofilaments and possibly other substrates. Myelination by Schwann cells sculpts the axon-altering functional architecture, electrical properties, and neuronal morphologies. 相似文献
13.
Changes in neurofilament transport coincide temporally with alterations in the caliber of axons in regenerating motor fibers 总被引:15,自引:7,他引:8 下载免费PDF全文
The delivery of neurofilaments via axonal transport has been proposed as an important mechanism for regulating axonal caliber. If this hypothesis is correct, alterations in axonal caliber should appear coincident with changes in the delivery of neurofilaments to the axon. The purpose of this study was to determine whether alterations in the caliber of axons in the proximal stumps of transected motor fibers precede, coincide with, or occur substantially later than changes in the delivery of neurofilaments via axonal transport. Between 3 d and 12 wk after crushing the sciatic nerves of 7-wk-old rats, lumbar motor neurons were labeled by the intraspinal injection of [35S]methionine. In neurons labeled between 3 d and 6 wk after axotomy, the relative amount of neurofilament protein in the slow component, as reflected by the ratio of the radioactivities of the 145-kD neurofilament protein to tubulin, was reduced to 30-40% of the control value. Moreover, as determined by immunoreactivity on blots, the amounts of neurofilament protein and tubulin in these nerve fibers were reduced fourfold and twofold, respectively. Thus, changes in the ratio of labeled neurofilament protein to tubulin correlated with comparable changes in the quantities of these proteins in nerve fibers. This decrease in the quantity of neurofilament proteins delivered to axons coincided temporally with reductions in axonal caliber. After regeneration occurred, the delivery of neurofilament proteins returned to pre-axotomy levels (i.e., 8 wk after axotomy), and caliber was restored with resumption of normal age-related radial growth of these axons. Thus, changes in axonal caliber coincided temporally with alterations in the delivery of neurofilament proteins. These results suggest that the majority of neurofilaments in these motor fibers continuously move in the anterograde direction as part of the slow component of axonal transport and that the transport of neurofilaments plays an important role in regulating the caliber of these axons. 相似文献
14.
《The Journal of cell biology》1993,121(2):375-386
We have analyzed the dynamics of neuronal intermediate filaments in living neurons by using the method of photobleaching of fluorescently- labeled neurofilament L protein and immunoelectron microscopy of incorporation sites of biotinylated neurofilament L protein. Low-light- level imaging and photobleaching of growing axons of mouse sensory neurons did not affect the rate of either axonal growth or the addition of intermediate filament structures at the axon terminal, suggesting that any perturbations caused by these optical methods would be minimal. After laser photobleaching, recovery of fluorescence did occur slowly with a recovery half-time of 40 min. Furthermore, we observed a more rapid fluorescence recovery in growing axons than in quiescent ones, indicating a growth-dependent regulation of the turnover rate. Incorporation sites of biotin-labeled neurofilament L protein were localized as numerous discrete sites along the axon, and they slowly elongated to become continuous arrays 24 h after injection. Collectively, these results indicate that neuronal intermediate filaments in growing axons turn over within the small area of the axoplasm possibly by the mechanism of lateral and segmental incorporation of new subunits. 相似文献
15.
Phosphorylation on carboxyl terminus domains of neurofilament proteins in retinal ganglion cell neurons in vivo: influences on regional neurofilament accumulation, interneurofilament spacing, and axon caliber 总被引:11,自引:4,他引:7 下载免费PDF全文
《The Journal of cell biology》1994,126(4):1031-1046
The high molecular weight subunits of neurofilaments, NF-H and NF-M, have distinctively long carboxyl-terminal domains that become highly phosphorylated after newly formed neurofilaments enter the axon. We have investigated the functions of this process in normal, unperturbed retinal ganglion cell neurons of mature mice. Using in vivo pulse labeling with [35S]methionine or [32P]orthophosphate and immunocytochemistry with monoclonal antibodies to phosphorylation- dependent neurofilament epitopes, we showed that NF-H and NF-M subunits of transported neurofilaments begin to attain a mature state of phosphorylation within a discrete, very proximal region along optic axons starting 150 microns from the eye. Ultrastructural morphometry of 1,700-2,500 optic axons at each of seven levels proximal or distal to this transition zone demonstrated a threefold expansion of axon caliber at the 150-microns level, which then remained constant distally. The numbers of neurofilaments nearly doubled between the 100- and 150- microns level and further increased a total of threefold by the 1,200- microns level. Microtubule numbers rose only 30-35%. The minimum spacing between neurofilaments also nearly doubled and the average spacing increased from 30 nm to 55 nm. These results show that carboxyl- terminal phosphorylation expands axon caliber by initiating the local accumulation of neurofilaments within axons as well as by increasing the obligatory lateral spacing between neurofilaments. Myelination, which also began at the 150-microns level, may be an important influence on these events because no local neurofilament accumulation or caliber expansion occurred along unmyelinated optic axons. These findings provide evidence that carboxyl-terminal phosphorylation triggers the radial extension of neurofilament sidearms and is a key regulatory influence on neurofilament transport and on the local formation of a stationary but dynamic axonal cytoskeletal network. 相似文献
16.
Ackerley S Grierson AJ Brownlees J Thornhill P Anderton BH Leigh PN Shaw CE Miller CC 《The Journal of cell biology》2000,150(1):165-176
Neurofilaments are transported through axons by slow axonal transport. Abnormal accumulations of neurofilaments are seen in several neurodegenerative diseases, and this suggests that neurofilament transport is defective. Excitotoxic mechanisms involving glutamate are believed to be part of the pathogenic process in some neurodegenerative diseases, but there is currently little evidence to link glutamate with neurofilament transport. We have used a novel technique involving transfection of the green fluorescent protein-tagged neurofilament middle chain to measure neurofilament transport in cultured neurons. Treatment of the cells with glutamate induces a slowing of neurofilament transport. Phosphorylation of the side-arm domains of neurofilaments has been associated with a slowing of neurofilament transport, and we show that glutamate causes increased phosphorylation of these domains in cell bodies. We also show that glutamate activates members of the mitogen-activated protein kinase family, and that these kinases will phosphorylate neurofilament side-arm domains. These results provide a molecular framework to link glutamate excitotoxicity with neurofilament accumulation seen in some neurodegenerative diseases. 相似文献
17.
The purpose of the present study was to investigate the participation of the motor proteins kinesin and dynein in axonal transport of neurofilaments (NF) in cultured dorsal root ganglia neurons. Therefore, we performed live-recording studies of the green fluorescent protein-tagged neurofilament M (GFP-NF-M) to assay transport processes in neurons. Co-localization studies revealed that GFP-NF-M was capable to build a functional NF network with other NF subunits, including phosphorylated heavy neurofilaments (NF-H-PH). Time-lapse recordings using confocal laser scanning microscopy exhibited fast transport of NF dots in anterograde and retrograde direction through a photobleached gap. Following microinjection of anti-kinesin antibodies or colchicine treatment an impairment of anterograde as well as retrograde NF transport was observed during live-recording experiments. In contrast, microinjection of anti-dynein antibodies only impaired retrograde transport of NF whereas the anterograde movement of GFP-NF-M was unaffected. Treatment of the cells with unspecific antibodies had no effect. 相似文献
18.
Brady ST 《Nature cell biology》2000,2(3):E43-E45
In neurons, cytoskeletal proteins are transported from where they are made - the cell body - along the axons, but it has long been disputed whether they are transported as subunits or polymers. A new analysis of neurofilament movement may help to resolve the controversy. 相似文献
19.
Differential turnover of phosphate groups on neurofilament subunits in mammalian neurons in vivo 总被引:11,自引:0,他引:11
The phosphorylation and dephosphorylation of specific proteins was demonstrated directly in the intact vertebrate nervous system in vivo. By exploiting the neurons' ability to segregate a select group of cytoskeletal proteins from most other phosphorylated constituents of the cell by axoplasmic transport, we were able to examine the dynamics of phosphate turnover on neurofilament proteins in mouse retinal ganglion cell neurons simultaneously labeled with [32P]orthophosphate and [3H]proline in vivo. Three [3H]proline-labeled neurofilament protein (NFP) subunits, designated H (160-200 kDa), M (135-145 kDa), and L (68-70 kDa), entered optic axons in a mole:mole ratio similar to that of isolated axonal neurofilaments, supporting the notion that newly synthesized NFPs are transported along axons as assembled neurofilaments. NFP subunits incorporated high levels of 32P before reaching axonal sites at the level of the optic nerve. As neurofilaments were transported along axons, however, many initially incorporated [32P]phosphate groups were removed. Loss of these phosphate groups occurred to a different extent on each subunit. A minimum of 50-60 and 35-40% of the labeled phosphate groups was removed in a 5-day period from the L and M subunits, respectively. By contrast, the H subunit exhibited relatively little or no phosphate turnover during the same period. Dephosphorylation of L in axons is accompanied by a decrease in its net state of phosphorylation; changes in the phosphorylation state of H and M, however, also reflect ongoing addition of phosphates to these polypeptides during axonal transport (Nixon, R.A., Lewis, S.E., and Marotta, C.A. (1986) J. Neurosci., in press). The possibility is raised that dynamic rearrangements of phosphate topography on NFPs represent a mechanism to coordinate interactions of neurofilaments with other proteins as these elements are transported and incorporated into the stationary cytoskeleton along retinal ganglion cell axons. 相似文献
20.
Defective Neurofilament Transport in Mouse Models of Amyotrophic Lateral Sclerosis: A Review 总被引:8,自引:0,他引:8
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. 相似文献