Slow Component Of Axonal Transport Research Articles

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve.

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study of this movement in situ was only possible using radioisotopic pulse-labeling, which permits analysis of axonal transport in whole nerves with a temporal resolution of days and a spatial resolution of millimeters. To study neurofilament transport in situ with higher temporal and spatial resolution, we developed a hThy1-paGFP-NFM transgenic mouse that expresses neurofilament protein M tagged with photoactivatable GFP in neurons. Here we describe fluorescence photoactivation pulse-escape and pulse-spread methods to analyze neurofilament transport in single myelinated axons of tibial nerves from these mice ex vivo. Isolated nerve segments are maintained on the microscope stage by perfusion with oxygenated saline and imaged by spinning disk confocal fluorescence microscopy. Violet light is used to activate the fluorescence in a short axonal window. The fluorescence in the activated and flanking regions is analyzed over time, permitting the study of neurofilament transport with temporal and spatial resolution on the order of minutesand microns, respectively. Mathematical modeling can be used to extract kinetic parameters of neurofilament transport including the velocity, directional bias and pausing behavior from the resulting data. The pulse-escape and pulse-spread methods can also be adapted to visualize neurofilament transport in other nerves. With the development of additional transgenic mice, these methods could also be used to image and analyze the axonal transport of other cytoskeletal and cytosolic proteins in axons.

Modeling the slowing of neurofilament transport along the mouse sciatic nerve

Neurofilaments are transported along axons in the slow component of axonal transport. The average rate of movement is generally quoted as several millimeters or tenths of a millimeter per day, but this rate is known to decrease while the neurofilaments are in transit due to spatial and temporal factors that are not understood. We have previously presented a stochastic model for neurofilament movement in vivo based on the transport kinetics of single neurofilaments observed by time-lapse fluorescence imaging in cultured neurons. The model took into account multiple velocity states and was only accessible through computational simulations. In simulations of the movement of a pulse of radiolabeled neurofilaments, this model generated a Gaussian wave which closely matched the experimental data. Here we present a simpler model with only three velocity states which is more amenable to analytical approaches. We show that the transport wave can be fully described by the mean and variance and we present analytical solutions for these cumulants in terms of the kinetic parameters of the model. We use the resulting expressions to examine the slowing of neurofilament transport in the mouse sciatic nerve. We show that the slowing is accompanied by an increase in the spread of the wave and that these changes are most readily explained by a change in the rate at which neurofilaments reverse their direction of movement. This suggests that the directionality of neurofilament transport in axons may be under spatial and/or temporal control and that alterations in the directionality of neurofilament transport could provide a mechanism for regulating the transport and distribution of these cytoskeletal polymers along axons.

Axonal transport of membranous and nonmembranous cargoes: a unified perspective.

Membranous and nonmembranous cargoes are transported along axons in the fast and slow components of axonal transport, respectively. Recent observations on the movement of cytoskeletal polymers in axons suggest that slow axonal transport is generated by fast motors and that the slow rate is due to rapid movements interrupted by prolonged pauses. This supports a unified perspective for fast and slow axonal transport based on rapid movements of diverse cargo structures that differ in the proportion of the time that they spend moving. A Flash feature (http://www.jcb.org/cgi/content/full/jcb.200212017/DC1) accompanies this Mini-Review.

Live-Cell Imaging of Slow Axonal Transport in Cultured Neurons

Cytoskeletal polymers and other cytosolic protein complexes are transported along axons in the slow components of axonal transport. Studies on the movement of neurofilaments and microtubules in the axons of cultured neurons indicate that these polymers actually move at fast rates and that the movements are also infrequent and highly asynchronous. These observations indicate that the slow rate of slow axonal transport is due to rapid movements interrupted by prolonged pauses which presents special challenges for studies on the mechanism of movement. This chapter describes the procedures that the author's laboratory has used to observe and analyze the movement of neurofilaments and microtubules in axons of cultured neurons from the superior cervical ganglia of neonatal rats. In particular, the author describes how to culture these neurons, how to transfect them by nuclear injection, and how to detect the rapid and infrequent movement of cytoskeletal polymers using time-lapse fluorescence imaging.

Rapid Movement of Microtubules in Axons

Cytoskeletal and cytosolic proteins are transported along axons in the slow components of axonal transport at average rates of about 0.002–0.1 μm/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 μm/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.

The slow axonal transport of the microtubule-associated protein tau and the transport rates of different isoforms and mutants in cultured neurons.

We demonstrate that the microtubule-associated protein tau, in the form of enhanced green fluorescent protein (EGFP) tau, is transported along axons of neurons in culture in the slow component of axonal transport with a speed comparable with that previously measured in vivo. It was demonstrated that the EGFP tag has no effect on transport characteristics, and the methodology enables slow transport rates of individual tau isoforms and tau mutants to be measured. We also expressed EGFP-tagged tau isoforms containing either three or four C-terminal repeats and zero or two N-terminal inserts in cultured neurons. No significant differences were found in the average rate of slow transport of the wild-type tau isoforms, suggesting that the exon 10 C-terminal repeat or the N-terminal inserts do not contain regions that play a significant regulatory role in axonal transport. Similarly, we found that missense mutations in tau have no noticeable effect on the rate of transport; hence their ability to cause neurodegeneration is by another mechanism other than that affecting the overall slow axonal transport of tau.

The Slow Axonal Transport of the Microtubule-Associated Protein Tau and the Transport Rates of Different Isoforms and Mutants in Cultured Neurons

We demonstrate that the microtubule-associated protein tau, in the form of enhanced green fluorescent protein (EGFP) tau, is transported along axons of neurons in culture in the slow component of axonal transport with a speed comparable with that previously measured in vivo. It was demonstrated that the EGFP tag has no effect on transport characteristics, and the methodology enables slow transport rates of individual tau isoforms and tau mutants to be measured. We also expressed EGFP-tagged tau isoforms containing either three or four C-terminal repeats and zero or two N-terminal inserts in cultured neurons. No significant differences were found in the average rate of slow transport of the wild-type tau isoforms, suggesting that the exon 10 C-terminal repeat or the N-terminal inserts do not contain regions that play a significant regulatory role in axonal transport. Similarly, we found that missense mutations in tau have no noticeable effect on the rate of transport; hence their ability to cause neurodegeneration is by another mechanism other than that affecting the overall slow axonal transport of tau.

Cytoplasmic dynein conversion at a crush injury in rat peripheral axons

Cytoplasmic dynein is a motor for retrograde axonal transport for movement of membranous organelles toward the neuronal cell body. However, cytoplasmic dynein is synthesized in the cell body and conveyed along the axon to nerve terminals. To characterize the axonal transport of cytoplasmic dynein in relation to synaptic vesicles and other membrane compartments, immunocytochemical and cytofluorimetric scanning analyses of crush-operated rat sciatic nerves were performed. Distal to the crush, the kinetics of dynein accumulation were consistent with its role in the retrograde transport of membranous organelles. During the initial 3 hr after crush, only small amounts of dynein-immunoreactive material accumulated proximal to the crush. This is consistent with metabolic labeling studies showing that most of the dynein moving in the anterograde direction is in the slow component of axonal transport. Thereafter, the rate of proximal accumulation of dynein increased, and by 8 hr postcrush a large amount of dynein immunoreactivity was observed. This accelerated accumulation may be due to recruitment of dynein from slow component b onto organelles proximal to the crush. Double labeling demonstrated that dynein immunoreactivity colocalized with synaptophysin, a transmembrane protein found in small, clear synaptic vesicles. In contrast, dynein immunoreactivity did not colocalize well with calcitonin gene-related peptide (CGRP), a peptide matrix marker for some large dense-cored vesicles. Finally, dynein immunoreactivity colocalized with the anterograde transport motor kinesin both proximal and distal to a crush, suggesting that kinesin may carry some dynein-containing membrane compartments during fast anterograde axonal transport. J. Neurosci. Res. 61:151–161, 2000. © 2000 Wiley-Liss, Inc.

Cytoplasmic dynein conversion at a crush injury in rat peripheral axons.

Cytoplasmic dynein is a motor for retrograde axonal transport for movement of membranous organelles toward the neuronal cell body. However, cytoplasmic dynein is synthesized in the cell body and conveyed along the axon to nerve terminals. To characterize the axonal transport of cytoplasmic dynein in relation to synaptic vesicles and other membrane compartments, immunocytochemical and cytofluorimetric scanning analyses of crush-operated rat sciatic nerves were performed. Distal to the crush, the kinetics of dynein accumulation were consistent with its role in the retrograde transport of membranous organelles. During the initial 3 hr after crush, only small amounts of dynein-immunoreactive material accumulated proximal to the crush. This is consistent with metabolic labeling studies showing that most of the dynein moving in the anterograde direction is in the slow component of axonal transport. Thereafter, the rate of proximal accumulation of dynein increased, and by 8 hr postcrush a large amount of dynein immunoreactivity was observed. This accelerated accumulation may be due to recruitment of dynein from slow component b onto organelles proximal to the crush. Double labeling demonstrated that dynein immunoreactivity colocalized with synaptophysin, a transmembrane protein found in small, clear synaptic vesicles. In contrast, dynein immunoreactivity did not colocalize well with calcitonin gene-related peptide (CGRP), a peptide matrix marker for some large dense-cored vesicles. Finally, dynein immunoreactivity colocalized with the anterograde transport motor kinesin both proximal and distal to a crush, suggesting that kinesin may carry some dynein-containing membrane compartments during fast anterograde axonal transport.

Characterization of NF-L and βIIΣ1-Spectrin Interaction in Live Cells

Neurofilaments (NFs) are neuron-specific intermediate filaments (IFs) composed of three different subunits, NF-L, NF-M, and NF-H. NFs move down the axon with the slow component of axonal transport, together with microtubules, microfilaments, and αII/βII-spectrin (nonerythroid spectrin or fodrin). It has been shown that αII/βII-spectrin is closely associated with NFs in vivo and that βII-spectrin subunit binds to NF-L filaments in vitro. In the present study we seek to elucidate the relationship between NF-L and βII-spectrin in vivo. We transiently transfected full-length NF-L and carboxyl-terminal deleted NF-L mutants in SW13 Cl.2 Vim− cells, which lack an endogenous IF network and express αII/βIIΣ1-spectrin. Double-immunofluorescence and electron microscopy studies showed that a large portion of βIIΣ1-spectrin colocalizes with the structures formed by NF-L proteins. We found a similar association between NF-L proteins and actin. However, coimmunoprecipitation experiments in transfected cells and the yeast two-hybrid system results failed to demonstrate a direct interaction of NF-L with βIIΣ1-spectrin in vivo. The presence of another protein that acts as a bridge between the membrane skeleton and neurofilaments or modulating their association may therefore be required.

Review : The Synthesis, Axonal Transport, and Phosphorylation of Neurofilaments Determine Axonal Caliber in Myelinated Nerve Fibers

Axonal diameter is the principal determinant of conduction velocity in myelinated nerve fibers, but, until recently, the factors that determine axonal diameter have not been understood. Recent studies indicate that neurofilaments (NFs), the principal intermediate (10-nm) filaments in neurons, are major intrinsic determinants of axonal caliber and have begun to elucidate the molecular mechanisms involved in NF deployment. The radial growth of myelinated axons during postnatal development reflects an increase in axonal NF content. The NF proteins are synthesized in the neuron cell body (soma) and transported somatofugally along axons in the slow component of axonal transport. The level of NF expression de termines the amount of NF protein transported in the slow component. In neurons with large axons, NF expression rises dramatically during neonatal development and is maintained at high steady-state levels as the NF content of axons increases during postnatal development. This increase in NF content, after NF expression has reached steady-state levels, appears to result from a progressive decline in the ve locity of NF transport, with increasing distance along nerve fibers (i.e., NFs enter a region of the axon faster than they leave). Phosphorylation of the NF proteins may regulate the spacing of axonal NFs and the velocity of NF transport. The Neuroscientist 1:76-83, 1995

Axonal transport of class II and III beta-tubulin: evidence that the slow component wave represents the movement of only a small fraction of the tubulin in mature motor axons.

Pulse-labeling studies demonstrate that tubulin synthesized in the neuron cell body (soma) moves somatofugally within the axon (at a rate of several millimeters per day) as a well-defined wave corresponding to the slow component of axonal transport. A major goal of the present study was to determine what proportion of the tubulin in mature motor axons is transported in this wave. Lumbar motor neurons in 9-wk-old rats were labeled by injecting [35S]methionine into the spinal cord 2 wk after motor axons were injured (axotomized) by crushing the sciatic nerve. Immunoprecipitation with mAbs which recognize either class II or III beta-tubulin were used to analyze the distributions of radioactivity in these isotypes in intact and axotomized motor fibers 5 d after labeling. We found that both isotypes were associated with the slow component wave, and that the leading edge of this wave was enriched in the class III isotype. Axotomy resulted in significant increases in the labeling and transport rates of both isotypes. Immunohistochemical examination of peripheral nerve fibers demonstrated that nearly all of the class II and III beta-tubulin in nerve fibers is located within axons. Although the amounts of radioactivity per millimeter of nerve in class II and III beta-tubulin were significantly greater in axotomized than in control nerves (with increases of +160% and +58%, respectively), immunoassay revealed no differences in the amounts of these isotypes in axotomized and control motor fibers. We consider several explanations for this paradox; these include the possibility that the total tubulin content is relatively insensitive to changes in the amount of tubulin transported in the slow component wave because this wave represents the movement of only a small fraction of the tubulin in these motor fibers.

Axonal transport kinetics and posttranslational modification of synapsin I in mouse retinal ganglion cells

Synapsin I is a neuron-specific phosphoprotein primarily localized at the presynaptic terminals, where it is thought to play an important role in the mechanisms involved in neurotransmitter release. Its interaction with cytoskeletal proteins and with small synaptic vesicles is regulated in vitro by phosphorylation by a calcium/calmodulin-dependent kinase. Here, we present the first evidence that, in the mouse retinal ganglion cells, synapsin I, moving along the axon with the slow component of axonal transport, is phosphorylated in vivo at both the head and tail regions. In addition, our data suggest that, after synapsin I has reached the nerve endings, the relative proportion of differently phosphorylated molecules of synapsin I changes, and that these changes lead to a decrease of the overall content of phosphorus. The more basic forms, here collectively referred to as beta-forms, become predominant at the terminals after 7 d postlabeling, when the bulk of transported synapsin I has entered the superior colliculus. Along the axon, phosphorylation could be functional in preventing synapsin I from forming, with actin, a dense meshwork that would restrict organelle movement. On the other hand, at the terminals, the dephosphorylation-phosphorylation of synapsin I may regulate the clustering of small synaptic vesicles and modulate neurotransmitter release by controlling the availability of small synaptic vesicles for exocytosis.

Cytotypic differences in the protein composition of the axonally transported cytoskeleton in mammalian neurons

Many of the structural and functional differences between axons are thought to reflect underlying differences in the biochemical composition and dynamic aspects of the axonal cytoskeleton and cytomatrix. In this study we investigated how the composition of the 2 slow components of axonal transport, SCa and SCb, which convey the cytoskeleton and cytomatrix, differs in axons that are structurally and functionally distinct. For this comparison we analyzed axons of retinal ganglion cells in the optic nerve (ON), axons of dorsal root ganglion (DRG) cells, and axons of ventral motor neurons (VMN) in adult rats. 35S-Methionine-labeled proteins transported with the peak of SCa and SCb were analyzed using high-resolution 2-dimensional polyacrylamide gels (2D-PAGE) and fluorography, and the amounts of major SCa and SCb proteins were quantified. The polypeptide composition of both SCa and SCb was found to be largely similar in DRG and VMN axons, but major qualitative as well as quantitative differences between these axons and ON axons were found. Notable among these were higher ratios of neurofilament protein to tubulin in SCa in DRG and VMN axons compared to ON axons, and significantly larger amounts of 2 microtubule-associated proteins relative to tubulin in SCa of ON axons than in both VMN and DRG axons. Tubulin was the major SCb protein in VMN and DRG axons, but it was not present in SCb in ON axons. Additionally, relatively larger amounts of 2 metabolic enzymes, creatine phosphokinase and nerve-specific enolase, were present in SCb in ON axons than in DRG or VMN axons. The results indicate that significant biochemical heterogeneity among different types of axons can be identified by examining the slow components of axonal transport.

Structural protein transport in elongating motor axons after sciatic nerve crush

In elongating motor axons of the rat sciatic nerve, the maximum outgrowth rate increased from 4.6 to 5.3 mm/d (5.3-6.1 X 10(-8) m/s) when a testing lesion of spinal nerves L4 and L5 was preceded 2 wk earlier by a conditioning lesion of the sciatic nerve. Axonal outgrowth was examined by measuring the transport of 35[S]methionine-labeled structural proteins (tubulin, actin, and neurofilament triplet) from "parent" axon stumps into "daughter" axon sprouts. Since these proteins are conveyed by the slow component of axonal transport at 1-5 mm/d (1.2-6.0 X 10(-8) m/s), the isotope was injected into the spinal cord 1 wk before the testing lesion. Nerves were removed 8 d after the testing lesion, sectioned into 3-mm segments, and homogenized; soluble proteins were separated by polyacrylamide gel electrophoresis. Fluorographs were used as templates to identify gel segments for removal, solubilization, and liquid scintillation counting. Distributions of mean radioactivity for tubulin, actin, and neurofilament triplet were plotted for animals receiving a conditioning vs sham-conditioning lesion. Greater amounts of tubulin and actin were transported into daughter axons in the conditioned group. Tubulin was mainly increased in axon shafts, whereas actin was mainly increased in axon tips. These findings suggest that the axonal transport of tubulin and actin governs the rate of elongation.

Changes in neurofilament transport coincide temporally with alterations in the caliber of axons in regenerating motor fibers.

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.

Slow axonal transport in acrylamide neuropathy: different abnormalities produced by single-dose and continuous administration

Alterations in axonal caliber and neurofilament content have been associated with altered neurofilament transport in several models of neurofibrillary degeneration. Acrylamide intoxication provides a prototype of distal axonal degeneration, the most frequent pattern of axonal pathology in human and experimental neurotoxic injury. Neurofibrillary changes are a variable and often minor aspect of the early pathological changes observed in acrylamide intoxication, and previous studies of slow axonal transport have produced conflicting results. In this study, we have correlated slow axonal transport, specifically neurofilament transport, with structural changes in the sciatic nerve complex of rats exposed to acrylamide. To study direct toxic effects of acrylamide, young rats were given a single dose of acrylamide (75 mg/kg, i.p.). A second group received daily injections of acrylamide at a lower dose (30 mg/kg, i.p.) in order to study animals with established acrylamide neuropathy. The slow component of axonal transport was labeled by intraspinal injections of [35S] methionine. Transport of individual slow component polypeptides was compared to profiles obtained from age-matched controls. Similarly intoxicated rats were perfused for morphometric and morphological studies. Results demonstrate that two different abnormalities of the slow component of axonal transport arise at different stages during the development of experimental acrylamide neuropathy. Both patterns of altered transport have structural correlates which reflect the changes in neurofilament transport. Following a single high dose, there was a modest retardation of the leading edge of the slow component. At this time, neurofilaments accumulated in proximal axons with formation of axonal swellings. During chronic administration, when distal axonal degeneration was present, the proportion of neurofilaments in the slow component was markedly reduced, and there was prominent loss of caliber in proximal axons. We suggest that these early changes represent a direct toxic effect of acrylamide on slow transport, whereas the later changes reflect reordering of slow transport as a neuronal response to toxin-induced axonal injury. This latter effect is of sufficient magnitude to obscure the acrylamide-induced retardation of slow transport.

Slow components of axonal transport: two cytoskeletal networks.

We have identified two slowly moving groups of axonally transported proteins in guinea pig retinal ganglion cell axons (4). The slowest group of proteins, designated slow component a (SCa), has a transport rate of 0.25 mm/d and consists of tubulin and neurofilament protein. The other slowly transported group of proteins, designated slow components b (SCb), has a transport rate of 2-3 mm/d and consists of many polypeptides, one of which is actin (4). Our analyses of the transport kinetics of the individual polypeptides of SCa and SCb indicate that (a) the polypeptides of SCa are transported coherently in the optic axons, (b) the polypeptides of SCb are also transported coherently but completely separately from the SCa polypeptides, and (c) the polypeptides of SCa differ completely from those comprising SCb. We relate these results to our general hypothesis that slow axonal transport represents the movements of structural complexes of proteins. Furthermore, it is proposed that SCa corresponds to the microtubule-neurofilament network, and that SCb represents the transport of the microfilament network together with the proteins complexed with microfilaments.

Characterization of the fast and slow components of axonal transport in retinal ganglion cells

AbstractThe constituent proteins of the fast (110–150 mm/day) and slow (1.5–2 mm/day) components of axonal transport in the retinal ganglion cells of the rabbit were investigated. The fast and slow components were labelled by intraocular injection of (3H)‐ and (14C)‐leucine, respectively.Subcellular fractionation of the optic nerve and tract and subsequent gel electrophoresis of the fractions showed that most of the soluble proteins moved with the slow phase of axonal transport, whereas only some of the soluble proteins were transported with the rapid phase.Extraction of the microsomal fraction with triton X‐100 resulted in the solubilization of highly labelled proteins belonging to the rapid phase. These proteins showed a relatively low electrophoretic mobility.

Components of fast and slow axonal transport in the goldfish optic nerve.

AbstractThe composition of the fast and slow components of axonal transport in the goldfish optic nerve was investigated, using specific radioactive precursors injected into the eye. Tritiated glucosamine and fucose label macromolecules, presumably glycoproteins, which are rapidly transported from the eye to the optic tectum. Material labeled with these precursors is not evident in the slowly transported component. Glucosamine and fucose incorporation are blocked when a protein synthesis inhibitor, acetoxycycloheximide, is injected into the eye concurrently with the precursors. As well as labeling macromolecules, 3H‐glucosamine and 3H‐N‐acetylmannosamine ( a precursor of sialic acids) also label rapidly‐transported chloroform‐methanol‐extractable material which may contain transported glycolipids.Two procedures were used to show that the slow component of axonal transport contains tubulin, a protein characteristic of the microtubules: Tracer doses of tritiated colchicine injected into the eye label a wave of radioactivity which moves 0.5 mm/day, the rate of slow axonal transport in the goldfish optic nerve. We believe this wave represents the movement of colchicine which is bound to colchicine‐binding protein moving in the slow component of axonal transport. Tritiated proline labels a slowly transported protein which is precipitated by vinblastine and has a mobility on polyacrylamide gels comparable to authentic tubulin. These results indicate that the fast and slow components of axonal transport each provide specific chemical substances to the nerve endings.