SLOW AXONAL TRANSPORT OF LABELLED PROTEINS IN SENSORY FIBRES OF RABBIT VAGUS NERVE

Both rapid (415 mm/day) and slow (24 mm/day) rates of axonal transport of proteins were found in sensory fibres of rabbit vagus nerve after injection of [³H]leucine into the nodose ganglion in vivo. The slow phase of transport was dependent on contact between the cell bodies and the nerve trunk, and did not continue under in vivro conditions. The results suggest some difference between the mechanisms of fast and slow transport.     

Ca2+- or Mg2+-Stimulated ATPase Activity in Bullfrog Spinal Nerve: Relation to Ca2+ Requirements for Fast Axonal Transport

Adenosine triphosphatase (ATPase) activity stimulated by Ca2+ or Mg2+ was characterized in spinal nerve and spinal sensory ganglion of bullfrog. Enzyme activity of homogenates from both sources reached a maximum at a 1-2 mM concentration of either cation, although the level of maximal activity in nerve trunks was approximately twice that in ganglia. Enzyme activation was not observed with 2 mM-Sr2+ or Ba2+. Co2+ or Mn2+, at 2 mM, depressed Ca2+ activation of the enzyme by 50-60% in nerve but had no inhibitory effect on ganglia activity. In intact spinal ganglion/spinal nerve preparations, incubated for 20 h in medium containing 0.2 mM-Co2+, no effect was detected on Ca2+/Mg2+ ATPase activity in ganglia or nerve trunks whereas fast axonal transport was inhibited by 80%. Incubation in medium containing 0.02 mM-Hg2+ depressed enzyme activity in ganglia by 64% and in nerve trunks by 44%, whereas fast transport was again inhibited by 80%. When only nerve trunks were exposed to these ions, Hg2+ but not Co2+ was observed to slow the rate of fast axonal transport. The divalent cation specificity of the Ca2+/Mg2+ ATPase activity is distinct from the ion specificities, determined in previous work, of the Ca2+ requirement during initiation of fast axonal transport in the soma, and of the Ca2+ requirement during translocation in the axon. Thus, previous observations of Ca2+-dependent events in fast axonal transport cannot be taken per se to suggest the involvement of Ca2+/Mg+ ATPase in the transport process.     

Changes in Protein Content of Goldfish Optic Nerve During Degeneration and Regeneration Following Nerve Crush

Abstract: After the goldfish optic nerve was crushed, the total amount of protein in the nerve decreased by about 45% within 1 week as the axons degenerated, began to recover between 2 and 5 weeks as axonal regeneration occurred, and had returned to nearly normal by 12 weeks. Corresponding changes in the relative amounts of some individual proteins were investigated by separating the proteins by two-dimensional gel electrophoresis and performing a quantitative analysis of the Coomassie Brilliant Blue staining patterns of the gels. In addition, labelling patterns showing incorporation of [³H]proline into individual proteins were examined to differentiate between locally synthesized proteins (presumably produced mainly by the glial cells) and axonal proteins carried by fast or slow axonal transport. Some prominent nerve proteins, ON1 and ON2 (50–55 kD, pI ～6), decreased to almost undetectable levels and then reappeared with a time course corresponding to the changes in total protein content of the nerve. Similar changes were seen in a protein we have designated NF (～130 kD, pI ～5.2). These three proteins, which were labelled in association with slow axonal transport, may be neurofilament constituents. Large decreases following optic nerve crush were also seen in the relative amounts of α- and β-tubulin, which suggests that they are localized mainly in the optic axons rather than the glial cells. Another group of proteins, W2, W3, and W4 (35–45 kD, pI 6.5–7.0), which showed a somewhat slower time course of disappearance and were intensely labelled in the local synthesis pattern, may be associated with myelin. A small number of proteins increased in relative amount following nerve crush. These included some, P1 and P2 (35–40 kD, pIs 6.1–6.2) and NT (～50 kD, pI ～5.5), that appeared to be synthesized by the glial cells. Increases were also seen in one axonal protein, B (～45 kD, pI ～4.5), that is carried by fast axonal transport, as well as in two axonal proteins, HA1 and HA2 (～60 and 65 kD respectively, pIs 4.5–5.0), that are carried mainly by slow axonal transport. Other proteins, including actin, that showed no net changes in relative amount (but presumably changed in absolute amount in direct proportion to the changes in total protein content of the nerve), are apparently distributed in both the neuronal and nonneuronal compartments of the nerve.     

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

The 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.     

TRANSPORT, TURNOVER AND DISTRIBUTION OF CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLIN-ESTERASE IN THE VAGUS AND HYPOGLOSSAL NERVES OF THE RABBIT

Abstract— The transport, distribution and turnover of choline O -acetyltransferase (ChAc, EC 2.3.1.6) and acetylcholinesterase (AChE, EC 3.1.1.7) in the vagus and hypoglossal nerves were studied in adult rabbits. The enzymes accumulated proximally and distally to single and double ligatures on both nerves and thus indicated both a proximo-distal and retrograde flow of the enzymes. Double ligature experiments indicated that only 5–20 per cent of the enzymes were mobile in the axon. The rate of accumulation of both enzymes above a single ligature corresponded to the slow rate of axonal flow provided that all the enzymes were mobile, but to an intermediate or fast flow if only a small part of the enzymes was transported. The distribution of ChAc along the hypoglossal neurons was studied and only 2 per cent of ChAc was confined to cell bodies, 42 per cent was localized to the main hypoglossal nerve trunks and 56 per cent to the preterminal axons and axon terminals in the tongue. The ratio of AChE to ChAc was about 3 in the hypoglossal nerve and 32 in the vagus nerve.
Transection of the hypoglossal nerve was followed by a decrease in the activity of ChAc in the hypoglossal nucleus and nerve and in the axons and their terminals in the tongue. The activity of AChE decreased in the hypoglossal nucleus and nerve but not in the tongue. The half-life of ChAc in preterminal axons and terminals of the hypoglossal nerve was estimated to be 16-21 days from the results obtained on transport, axotomy and distribution of the enzyme. Intracisternal injection of colchicine inhibited the cellulifugal transport of both enzymes and led to an increase in enzyme activity in the hypoglossal nucleus.     

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 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.     

Changes in the axonal protein transport under conditions of elevated functional activity of the neurons

The prolonged nonintensive physical activity by swimming without load (12 +/- 2 h.) has no effect on the overall amount of fast and slow transported proteins of transport velocity in rat central and peripheral sensory fibres of the sciatic nerve. However, the rate of fast axonal transport in the motor fibres decreases by 18% and the amount of proteins by a factor of 2 as compared with control. The rate of slow axonal transport does not change, but the mean level of transported labeled proteins decreases by 1.9 times. The relatively short-term but more intensive activity (swimming with the load during 60 +/- 10 min.) provokes an increase of the rate by 10% and the overall amount of fast transported proteins by 2 times. The rest of the animals during 6 h. returns the above parameters to control values. A suggestion is made that the rate and the amount of transported proteins depend on the variations in functional state of the neurons and their axons.     

Axonal Transport in the Garfish Optic Nerve: Comparison with the Olfactory System

Fast and slow axonal transports were studied in the optic nerve of the garfish and compared with previous studies on the olfactory nerve. The composition of fast-transport proteins was very similar in the two nerves. Although the velocity of fast transport was slightly lower in the optic nerve, there was a linear increase in velocity with temperature in both nerves. As in the olfactory nerve, only a single wave of slow-transport protein radioactivity moves along the nerve. The velocity of slow transport also increased linearly with temperature, but the coefficient was less than in the olfactory system. The composition of slow transport in the optic nerve was significantly different from that in the olfactory nerve, a finding reflecting the different cytoskeletal constituents of the two types of axons. The slow wave could be differentiated into several subcomponents, with the order of velocities being a 105-kilodalton protein and actin greater than tubulins and clathrin greater than fodrin much greater than neurofilaments. It can be concluded that the temperature dependence of fast and slow axonal transport in different nerves reflects the influence of temperature on the individual polypeptides constituting the various transport phases. The garfish optic nerve preparation may be advantageous for studies of axonal transport in retinal ganglion cell axons, because its great length avoids the complications of having to study transport in the optic tract or in material accumulating at the tectum.     

EFFECTS OF COLCHICINE ON AXONAL TRANSPORT IN PERIPHERAL NERVES

—Colchicine injected intracisternally markedly inhibited the rapid migration (300-400 mm/day) of labelled proteins in the hypoglossal and vagus nerve of the rabbit. The transport of acetylcholinesterase (EC 3.1.1.7) and choline acetyltransferase (EC 2.3.1.6) previously shown to move with the slow (5-26 mm/day) phase of axoplasmic transport in these nerves, was only partially blocked. In view of this differential effect on axonal flow, we suggest that the neurotubules, on which colchicine acts preferentially, are primarily involved in the rapid (300-400 mm/day) axoplasmic flow. After local injection of colchicine into the nerves both the rapidly migrating labelled proteins and the enzymes (AChE and ChAc) accumulated above the site of injection to the same degree as they accumulate above a nerve ligation. Since this blockage of enzyme transport occurred after concentrations of colchicine much higher than those used for intracisternal injections these findings after local injection may represent more severe effects on axonal transport systems.     

Cellular Origin and Biosynthesis of Rat Optic Nerve Proteins: A Two-Dimensional Gel Analysis

High resolution 2DGE (two-dimensional gel electrophoresis) was used to characterize neuronal and glial proteins of the rat optic nerve, to examine the phases of intraaxonal transport with which the neuronal proteins are associated, and to identify the ribosomal populations on which these proteins are synthesized. Neuronal proteins synthesized in the retinal ganglion cells were identified by injecting the eye with L-[35S]methionine, followed by 2DGE analysis of fast and slow axonally transported proteins in particulate and soluble fractions. Proteins synthesized by the glial cells were labeled by incubating isolated optic nerves in the presence of L-[35S]methionine and then analyzed by 2DGE. A number of differences were seen between filamentous proteins of neurons and glia. Most strikingly, proteins in the alpha- and beta-tubulin region of the 2D gels of glial proteins were distinctly different than was observed for axonal proteins. As expected, neurons but not glia expressed neurofilament proteins, which appeared among the slow axonally transported proteins in the particulate fraction; significant amounts of the glial filamentous protein, GFA, were also labeled under these conditions, which may have been due to transfer of amino acids from the axon to the glial compartment. The fast axonally transported proteins contained relatively large amounts of high-molecular-weight acidic proteins, two of which were shown to comigrate (on 2DGE) with proteins synthesized by rat CNS rough microsomes; this finding suggests that rough endoplasmic reticulum may be a major site of synthesis for fast transported proteins. In contrast, the free polysome population was shown to synthesize the principal components of slow axonal transport, including tubulin subunits, actin, and neurofilament proteins.     

Axoplasmic transport in the crayfish nerve cord. The role of fibrillar constituents of neurons

(a) Axoplasmic transport of tritium-labeled proteins in crayfish nerve cord was confirmed at a slow rate of 1 mm/day. A second proteinaceous component which moves at a rate of 10 mm/day was also detected. Radioautography and biochemical analysis indicate that proteins migrating at these velocities have a perikaryal origin and move caudad within axons as sharply defined peaks. (b) Evidence is presented for the blockage of the slow and the fast movement of proteins by intraganglionic injection of the anti-mitotic agent vinblastine sulfate (0.1 mM). (c) Electron microscope observations of vinblastine-treated ganglia revealed a reduction in the number of axonal microtubules and the formation of intracellular aggregates presumably composed of microtubular protein. (d) These findings would be compatible with the involvement of microtubules in both slow and fast axoplasmic transport. However, the block induced by vinblastine was detected in regions of the cord (up to 10 mm away from the injection site) where the number and morphology of microtubules appeared unaltered. In addition, axons showing effects of vinblastine occasionally contained mitochondria with remarkably dense and thickened membranes. (e) In association with the surfaces of axonal microtubules are lateral filamentous elements (40–80 A in diameter) which also showed vinblastine-induced alterations. Our observations indicate that such filiform structures, associated with microtubules, may be a necessary component in the transport mechanism(s).     

Microtubule stability along mammalian peripheral nerves

Microtubule (MT) number, axonal area, and MT density were examined in unmyelinated axons of rat cervical vagus nerve. Study of nerve regions proximal (1-5 mm) and distal (35-40 mm) to the nodosum ganglion in controls (incubation at 37 degrees C for 1 h) showed that the number of MT per axon is significantly less in distal than in proximal nerve regions. Cooling (incubation at 0 degree C for 1 h) caused a significant reduction in the number of MT per axon in both nerve regions. The unmyelinated axons from both nerve regions showed a comparable reduction in MT number by cooling, indicating that axonal MT stability to cold was not significantly different between these two nerve regions. In these nerves no detectable changes were found in cross-axonal area of unmyelinated axons between distal and proximal nerve regions. In another experimental series, in distal nerve regions (35-40 mm from the nodosum ganglion) the number of MT was not further reduced in nerves incubated at 0 degree C by increasing the incubation time. Similar results were obtained from colchicine treated nerves (incubation at 37 degrees C, with 10 mM colchicine for 1 and 2 h). Distal nerve regions (35-40 mm from the nodosum ganglion) showed a similar reduction in the number of MT per axon when nerves were incubated at 0 degree C or with colchicine, suggesting that this drug, as well as cold, may be affecting a similar population of axonal MT, i.e., MT susceptible to anti-MT agents. These results indicate that approximately one-half of the axonal MT are stable to cold as well as to colchicine in rat unmyelinated axons.     

Anterograde Components of Axonal Transport in Motor and Sensory Nerves in Experimental 2,5-Hexanedione Neuropathy

Anterograde slow and fast axonal transport was examined in rats intoxicated with 2,5-hexanedione (1 g/kg/week) for 8 weeks. Distribution of radioactivity was measured in 3-mm segments of the sciatic nerve after labelling of proteins with [35S]methionine or [3H]leucine and glycoproteins with [3H]fucose. The axonal transport of the anterograde slow components was examined after 25 (SCa) and 10 days (SCb), in motor and sensory nerves. SCa showed an increased transport velocity in motor (1.25 +/- 0.08 mm/day versus 1.01 +/- 0.05 mm/day) and in sensory nerves (1.21 +/- 0.13 mm/day versus 1.06 +/- 0.07 mm/day). The relative amount of labelled protein in the SCa wave in both fiber systems was also increased. SCb showed unchanged transport velocity in motor as well as in sensory nerves, whereas the amount of label was decreased in the motor system. Anterograde fast transport in motor nerves was examined after intervals of 3 and 5 h, whereas intervals of 2 and 4 h were used for sensory nerves. Velocities and amounts of labelled proteins of the anterograde fast component remained normal. We suggest that the increase in protein transport in SCa reflects axonal regeneration.     

Inhibition of axonal transport in vitro in frog sciatic nerves by chlorpromazine and lidocaine

Summary The effects of chlorpromazine hydrochloride (CPZ HCl) and prochlorperazin-metansulfonate (PCPZ) on the fast axonal transport of labelled proteins were examined in vitro in a peripheral frog nerve.A 0.1 mM concentration of CPZ HCl and PCPZ reduced the amount of transported proteins by more than 50 per cent. An almost complete block was obtained with a 0.5 mM concentration of these two drugs. The lower concentration hardly affected the protein synthesis. The transport inhibiting effect of 0.1 mM of the drugs was reversible but not that of the higher concentration.The number of microtubuli was strongly decreased and the number of filaments increased at the transport inhibiting concentrations. The ultrastructural changes induced by 0.1 mM of the phenothiazine tranquilizer were largely reversible. The local anesthetics lidocaine (18.3 mM) and tetracaine (3.3 mM) both caused similar changes, i.e. a reduction in the number of microtubuli. No ultrastructural effects were observed after treatment with 1 mM ouabain. These three drugs are known to block the axonal flow in the present system at the above mentioned concentrations.The biochemical and ultrastructural results are discussed in relation to those induced by other drugs affecting axonal transport.The present work was supported by grants from Statens Naturvetenskapliga Forskningsråd (No. 2535-8), C.-B. Nathorsts Vetenskapliga och Allmännyttiga Stiftelser, the Swedish Medical Research Council (B73-12X-2543-05B), H. Hierta's Stiftelse and W. och M. Lundgrens Stiftelse. Thanks are due to Mrs B. Egnér, Mrs E. Fjällstedt, Mrs. E. Norström and Mrs U. Svedin for expert technical assistance.     

Melanocortins and fast axonal transport in intact and regenerating sciatic nerve

The effect of ACTH/MSH peptides on fast axonal transport along intact or regenerating sciatic nerve was examined following injection of tritiated leucine into the rat lumbar spinal cord. The rate of fast axonal transport was not significantly changed by treatment with ACTH/MSH(4-10), the ACTH(4-9) analog ORG 2766, hypophysectomy, or adrenalectomy. Fast axonal transport was unchanged in regenerating nerves and in regenerating, ACTH(4-10)-treated nerves. However, treatment with ORG 2766 in dosages of either 1 or 10 micrograms/kg/day IP for seven days significantly reduced (62% and 64%, respectively) the crest height of the fast axonal transport curve of intact sciatic nerve. The results suggest that the reported peptide-induced enhancement of nerve regeneration is not due to changes in the rate of fast axonal transport.     

Axotomy accelerates slow component b of axonal transport.

Because the integrity of an axon depends on the supply of proteins synthesized in the cell body, we examined the effect of axotomy on the transport of structural proteins in rat motor axons, and the effect of altered transport on the rate of outgrowth after a subsequent testing axotomy. To examine the axonal transport of structural proteins, we labeled newly synthesized proteins with 35S-methionine 7 days after a "conditioning" lesion of the sciatic nerve, and removed the nerve 7-21 days later for SDS-PAGE. Tubulin, actin, calmodulin, and the 68-kD light neurofilament protein (NF-L) were identified by fluorography and removed for liquid scintillation counting. The fastest moving structural proteins were carried by slow component b (SCb) of axonal transport, which advanced 20% faster in conditioned axons: 4.2 versus 3.5 mm/day (p less than 0.01). NF-L was not accelerated, indicating that the motor for subcomponent a (SCa) of slow axonal transport was unaffected by axotomy. To measure outgrowth distances, the testing lesions was made 7 days after the conditioning lesion, and growth cones were located by the fast transport method 3 or 9 days later. The regression analysis of outgrowth distance on time showed that sprouts elongated 25% faster in conditioned axons: 4.0 versus 3.2 mm/day (p less than 0.001). These accelerated sprouts were formed too far from the spinal cord to contain SCb proteins that were synthesized after axotomy. Because the rate of outgrowth correlated closely with the rate of SCb in outgrowing sprouts (McQuarrie and Jacob, J. Comp. Neurol. 305:139-147, 1991), we conclude that SCb is accelerated throughout the length of the axon by 7 days after axotomy.     

Kinesin‐1/Hsc70‐dependent mechanism of slow axonal transport and its relation to fast axonal transport

Cytoplasmic protein transport in axons (‘slow axonal transport’) is essential for neuronal homeostasis, and involves Kinesin‐1, the same motor for membranous organelle transport (‘fast axonal transport’). However, both molecular mechanisms of slow axonal transport and difference in usage of Kinesin‐1 between slow and fast axonal transport have been elusive. Here, we show that slow axonal transport depends on the interaction between the DnaJ‐like domain of the kinesin light chain in the Kinesin‐1 motor complex and Hsc70, scaffolding between cytoplasmic proteins and Kinesin‐1. The domain is within the tetratricopeptide repeat, which can bind to membranous organelles, and competitive perturbation of the domain in squid giant axons disrupted cytoplasmic protein transport and reinforced membranous organelle transport, indicating that this domain might have a function as a switchover system between slow and fast transport by Hsc70. Transgenic mice overexpressing a dominant‐negative form of the domain showed delayed slow transport, accelerated fast transport and optic axonopathy. These findings provide a basis for the regulatory mechanism of intracellular transport and its intriguing implication in neuronal dysfunction.     

RETROGRADE AXONAL TRANSPORT OF RAPIDLY MIGRATING LABELLED PROTEINS AND GLYCOPROTEINS IN REGENERATING PERIPHERAL NERVES

Abstract— The redistribution of rapidly migrating [³H]leucine-labelled proteins and [³H]fucose-labelled glycoproteins was studied in ligated regenerating hypoglossal and vagus nerves of the rabbit. When regenerating and contralateral hypoglossal nerves were ligated 16 h after labelling of the nerve cell bodies, rapidly migrating proteins and glycoproteins accumulated distal to the ligatures indicating a rapid retrograde transport from the peripheral parts of the nerves within 6 h. The retrograde accumulation of both proteins and glycoproteins was greater on the regenerating side than on the contralateral side at both 1 and 5 weeks after a nerve crush. Labelled proteins and glycoproteins also accumulated proximal to the ligatures, indicating a delayed rapid anterograde phase of axonal transport. The accumulation of this phase was also greater on the regenerating side 1 week after a nerve crush for both labelled proteins and glycoproteins. One week after a crush of the cervical vagus nerve, rapidly migrating proteins and glycoproteins redistributed between he crush zone and a proximal ligature applied 16 h after labelling of the nerve cell bodies. A retrograde accumulation occurred distal to the ligature within 6 h, indicating a rapid retrograde transport from the crush zone.     

RETROGRADE AXONAL TRANSPORT OF RAPIDLY MIGRATING PROTEINS IN THE VAGUS AND HYPOGLOSSAL NERVES OF THE RABBIT

—The redistribution of rapidly migrating [³H]leucine-labelled proteins was studied using double ligatures applied to the vagus nerve and single ligatures, applied to the hypoglossal nerves. Rapidly migrating proteins accumulating for 16 h proximal to a distal ligature of the cervical vagus redistributed to give a retrograde accumulation distal to a second ligature. Within 6 h a substantial redistribution occurred indicating a rapid retrograde transport. After 21 h there was a further accumulation with 70 per cent of the labelled material accumulating at the distal end of the isolated nerve segment and 16 per cent accumulating at the proximal end. It was shown that about a half of the retrograde accumulation was dependent on the distal accumulation zone. Rapidly migrating proteins accumulated distal to a ligature applied to the hypoglossal nerve 16 h after labelling of the nerve cell bodies indicating that a rapid retrograde transport of labelled macromolecules occurs from the peripheral parts of the nerve in the tongue. Labelled proteins accumulated proximal to ligatures and transections of both the hypoglossal and vagus nerve when applied 16 h after labelling of the nerve cell bodies, indicating the presence of axonal proteins, migrating at a rate of transport intermediate to that of rapidly and slowly migrating proteins.     

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

The 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:

• (a) 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.
• (b) 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.