Early structural changes in the axoplasmic cytoskeleton after axotomy studied by cryofixation

Summary Alterations in the cytoskeleton were studied in the axoplasm of neurites at the tips of proximal stumps of transected chicken sciatic nerves. The studies were carried out using cryofixation with a nitrogen-cooled propane jet. The most immediate effect is the almost complete disassembly of axoplasmic microtubules. This consequently causes the axonal transport of membrane-bounded organelles to cease and results in an accumulation of mitochondria and vesicles of the smooth endoplasmic reticulum. The neurofilament network is partially disorganized. Neurofilaments become shorter and fragmented, and are linked by a large number of anastomosed cross-linkers. The neurofilaments become newly aligned to the axis of the axoplasm and are of normal length 48–72 h after the transsection. At this stage the newly formed neurofilament bundles are in close proximity to the anastomosed cisternae and profiles of the smooth endoplasmic reticulum. The axonal sprouts always show a normally organized cytoskeletal network. These studies support the idea that the rapid remodelling of the neurofilament network is apparently a local event, not dependent on the slow transport of cytoskeletal materials to the tip of the proximal stump. The repair of the degraded cytoskeleton may be in accordance with the function of the endoplasmic reticulum as Ca²⁺-sequestering membrane system, which may be involved in restoring the physiological conditions of the axoplasm.     

Reorganization of axoplasmic organelles following beta, beta'- iminodipropionitrile administration

beta, beta'-Iminodipropionitrile (IDPN), a synthetic compound that selectively impairs slow axonal transport, produced a rearrangement of the axonal cytoskeleton, smooth endoplasmic reticulum, and mitochondria. Immunoperoxidase staining using an antiserum to the 68,000-dalton neurofilament subunit demonstrated a displacement of neurofilaments toward the periphery of the axons of IDPN-treated rats. This change occurred simultaneously along the entire length of the sciatic nerve. Ultrastructural morphometry of the axonal organelles confirmed the peripheral relocation of neurofilaments and also showed a displacement of microtubules, smooth endoplasmic reticulum, and mitochondria to the center of the axons. The overall density of axonal mitochondria was increased, whereas those of other organelles were not significantly changed. Axons were reduced in size by 10--24%, the large axons being more affected than the small ones. The observed rearrangement of axonal organelles may be due to an effect of IDPN on microtubule-neurofilament interactions, which could in turn explain the impairment of the slow transport. Axons in IDPN intoxication are a useful model to study the organization of the axoplasm and the mechanism of axonal transport.     

Redistribution of proteins of fast axonal transport following administration of beta,beta'-iminodipropionitrile: a quantitative autoradiographic study

Beta,beta'-iminodipropionitrile (IDPN) produces a rearrangement of axoplasmic organelles with displacement of microtubules, smooth endoplasmic reticulum, and mitochondria toward the center and of neurofilaments toward the periphery of the axon, whereas the rate of the fast component of axonal transport is unchanged. Separation of microtubules and neurofilaments makes the IDPN axons an excellent model for study of the role of these two organelles in axonal transport. The cross-sectional distribution of [3H]-labeled proteins moving with the front of the fast transport was analyzed by quantitative electron microscopic autoradiography in sciatic nerves of IDPN-treated and control rats, 6 h after injection of a 1:1 mixture of [3H]-proline and [3H]-lysine into lumbar ventral horns. In IDPN axons most of the transported [3H] proteins were located in the central region with microtubules, smooth endoplasmic reticulum and mitochondria, whereas few or none were in the periphery with neurofilaments. In control axons the [3H]-labeled proteins were uniformly distributed within the axoplasm. It is concluded that in fast axonal transport: (a) neurofilaments play no primary role; (b) the normal architecture of the axonal cytoskeleton and the normal cross-sectional distribution of transported materials are not indispensable for the maintenance of a normal rate of transport. The present findings are consistent with the models of fast transport that envision microtubules as the key organelles in providing directionality and propulsive force to the fast component of axonal transport.     

Two classes of actin microfilaments are associated with the inner cytoskeleton of axons

The distribution and length of actin microfilaments (MF) was determined in axoplasm extruded from the giant axons of the squid (Loligo pealeii). Extruded axoplasm that was separated from the axonal cortex contains approximately 92% of the total axonal actin, and 60% of this actin is polymerized (Morris, J., and R. Lasek. 1984. J. Cell Biol. 98:2064-2076). Localization of MF with rhodamine-phalloidin indicated that the MF were organized in fine columns oriented longitudinally within the axoplasm. In the electron microscope, MF were surrounded by a dense matrix and they were associated with the microtubule domains of the axoplasm. The surrounding matrix tended to obscure the MF which may explain why MF have rarely been recognized before in the inner regions of the axon. The axoplasmic MF are relatively short (number average length of 0.55 micron). Length measurements of MF prepared either in the presence or absence of the actin-filament stabilizing drug phalloidin indicate that axoplasm contains two populations of MF: stable MF (number average length of 0.79 micron) and metastable MF (number average length of 0.41 micron). Although individual axonal MF are much shorter than axonal microtubules, the combined length of the total MF is twice that of the total microtubules. Apparently, these numerous short MF have an important structural role in the architecture of the inner axonal cytoskeleton.     

A Stochastic Multiscale Model That Explains the Segregation of Axonal Microtubules and Neurofilaments in Neurological Diseases

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.     

Localization of synapsin I in normal fibers and regenerating axonal sprouts of the rat sciatic nerve

The localization of synapsin I, a synaptic vesicle-associated protein, was investigated immunocytochemically in normal nerve fibers and regenerating axonal sprouts following crush-injuries to the rat sciatic nerve. In normal myelinated axons, weak synapsin I immunoreactivity was found in the axoplasmic/smooth endoplasmic domains, but not in the cytoskeletal domains comprising neurofilaments and microtubules. In non-myelinated axons without dense cytoskeletal structures, moderate immunoreactivity was distributed diffusely throughout the axoplasm. In the crush-injured nerves, intense synapsin I immunoreactivity was demonstrated by light microscopy in early regenerating sprouts emerging from nodes of Ranvier. These nodal sprouts subsequently elongated as regenerating axons through the space between the basal lamina and the myelin sheath (or Schwann cell plasma membrane). Intense synapsin I immunoreactivity was also found in the growth cones of such long regenerating axons. Electron microscopy revealed that synapsin I immunoreactivity was associated mainly with vesicular organelles in the nodal sprouts and growth cones of regenerating axons. Long regenerating axons exhibited no synapsin I immunoreactivity in the shaft, which contained an abundance of neurofilaments. However, vesicle accumulations remaining in the periphery of the shaft still exhibited intense synapsin I immunoreactivity. Thus, it can be concluded that synapsin I is localized at especially high density in the domains comprising vesicular organelles, which are characteristic of early nodal sprouts, as well as in growth cones of regenerating axons. These findings, together with the proposed functions of synapsin I investigated in other studies, suggest that synapsin I may play important roles in vesicular dynamics including the translocation of vesicles to the plasma membrane in sprouts and growth cones of regenerating axons.     

The cytoskeleton of cryofixed Purkinje cells of the chicken cerebellum

Summary Cerebella of 3- to 6-week-old chickens were cryofixed in a nitrogen-cooled propane jet, deep-etched and rotary-shadowed. The use of a brief perfusion of 0.32 M sucrose improved the quality of the cryofixation and allowed the study of the deeper layers of the cerebellar cortex. It is reported that the cytoskeleton of the Purkinje cells (PC) shows distinct domains and composition of filamentous structures in the different regions of the cell cytoplasm, such as the perikaryon, the cytoplasm of dendrites and the axoplasm. The perikaryon is occupied by a meshwork of fine filaments, 4–7 nm in diameter, that extends from the nuclear outer membrane to the cell membrane. In this zone the cell organelles (e.g., endoplasmic reticulum, mitochondria) adopt a circular arrangement around the nucleus. All structures are anchored by microfilaments to the cytoplasmic network. The dendrites show a dense cytoplasmic network including bundles of microtubules, neurofilaments and microfilaments. Numerous aggregated globular components are attached to this cytoskeleton. The cytoskeleton of the dendritic spines shows axially oriented 10-nm bundles of filaments, which are interconnected and anchored also to the cell membrane and the components of the agranular endoplasmic reticulum by cross-linkers. As described in peripheral nerves, the axoplasm of axons in the central nervous system exhibits predominantly neurofilaments and microtubules aligned along the axis of the neuntes in a three-dimensional arrangement and interconnected by cross-linker filaments and filamentous structures.     

EFFECTS OF CALCIUM ION CONCENTRATION ON THE DEGENERATION OF AMPUTATED AXONS IN TISSUE CULTURE

Light and electron microscope studies were conducted on the nature of the degenerative changes in amputated nerve fibers of cultured rat sensory ganglia and on the effects of media with differing calcium concentrations upon these changes. With glucose-enriched Eagle's media (MEM) containing 1.6 mM calcium, the amputated myelinated and unmyelinated axons undergo a progressive granular disintegration of their axoplasm with collapse and fragmentation of myelin sheaths between 6 and 24 h after transection. With MEM containing only 25–50 µM calcium, the granular axoplasmic degeneration does not occur in transected fibers and they retain their longitudinal continuity and segmental myelin ensheathment for at least 48 h. Addition of 6 mM EGTA to MEM (reducing the estimated Ca⁺⁺ below 0.3 µM) results in the structural preservation of both microtubules and neurofilaments within transected axons. A transient focal swelling of amputated axons occurs, however, in cultures with normal and reduced calcium. These observations suggest that an alteration in the permeability of the axolemma is a crucial initiating event leading to axonal degenerative changes distal to nerve transection. The loss of microtubules and neurofilaments and the associated granular alterations of the axoplasm in transected fibers appears to result from the influx of calcium into the axoplasm.     

K+-selective microelectrode study of internally dialyzed squid giant axons.

Intracellular potassium activity, (aK)i, and axoplasmic K+ concentration, [K+]i, were measured by means of K+-selective microelectrodes and atomic absorption spectroscopy, respectively, in squid giant axons dialyzed with K+-free dialysis solution and bathed in K+-free artificial sea water. (aK)i measurements indicated that axoplasmic free K+ could be depleted by dialysis, whereas [K+]i measurements on axoplasm extruded from these axons suggest substantial retention of K+ (15.5 +/- 1.7 mmol/kg axoplasm K+; n = 9). In comparison, [K+]i in axoplasm extruded from freshly dissected axons was 330 +/- 16 mmol/kg axoplasm (n = 6). These data suggest that approximately 5% of the axoplasmic K+ ions are not easily removed by dialysis and that these ions are either bound to macromolecular sites or sequestered into membrane-enclosed organelles.     

Ultrastructural effects of colchicine on perikarya and initial segment of rat retinal ganglion cells.

Our ultrastructural study was focused on the perikaryal region and initial segment of the axon of rat retinal ganglion cells in controls and after intraocular injections of colchicine. In control rats that region contained, among other organelles, elements of the Golgi complex and, close to them, short isolated microtubules oriented preferentially toward the axon where they funnel and aggregate in bundles. One day after sufficient doses of colchicine to inhibit axoplasmic transport (2-20 micrograms) these cytoplasmic microtubules were absent, whereas some axonal microtubules were still present but reduced in number. In addition, colchicine induced an altered distribution of organelles, leaving empty spaces in the periphery and most organelles concentrated in the perinuclear region, especially around Golgi elements where numerous vesicles and tubules accumulate at the trans face of Golgi elements. These results suggest that the vesicles that leave the Golgi and have been directed towards axoplasmic transport may need the cytoplasmic microtubules located between Golgi elements and the axonal initial segments to reach the axon.     

Association of actin filaments with axonal microtubule tracts

Axoplasmic organelles move on actin as well as microtubules in vitro and axons contain a large amount of actin, but little is known about the organization and distribution of actin filaments within the axon. Here we undertake to define the relationship of the microtubule bundles typically found in axons to actin filaments by applying three microscopic techniques: laser-scanning confocal microscopy of immuno-labeled squid axoplasm; electronmicroscopy of conventionally prepared thin sections; and electronmicroscopy of touch preparations-a thin layer of axoplasm transferred to a specimen grid and negatively stained. Light microscopy shows that longitudinal actin filaments are abundant and usually coincide with longitudinal microtubule bundles. Electron microscopy shows that microfilaments are interwoven with the longitudinal bundles of microtubules. These bundles maintain their integrity when neurofilaments are extracted. Some, though not all microfilaments decorate with the S1 fragment of myosin, and some also act as nucleation sites for polymerization of exogenous actin, and hence are definitively identified as actin filaments. These actin filaments range in minimum length from 0.5 to 1.5 µm with some at least as long as 3.5 µm. We conclude that the microtubule-based tracks for fast organelle transport also include actin filaments. These actin filaments are sufficiently long and abundant to be ancillary or supportive of fast transport along microtubules within bundles, or to extend transport outside of the bundle. These actin filaments could also be essential for maintaining the structural integrity of the microtubule bundles.     

Organelle, bead, and microtubule translocations promoted by soluble factors from the squid giant axon

A reconstituted system for examining directed organelle movements along purified microtubules has been developed. Axoplasm from the squid giant axon was separated into soluble supernatant and organelle-enriched fractions. Movement of axoplasmic organelles along MAP-free microtubules occurred consistently only after addition of axoplasmic supernatant and ATP. The velocity of such organelle movement (1.6 micron/sec) was the same as in dissociated axoplasm. The axoplasmic supernatant also supported movement of microtubules along a glass surface and movement of carboxylated latex beads along microtubules at 0.5 micron/sec. The direction of microtubule movement on glass was opposite to that of organelle and bead movement on microtubules. The factors supporting movements of microtubules, beads, and organelles were sensitive to heat, trypsin, AMP-PNP and 100 microM vanadate. All of these movements may be driven by a single, soluble ATPase that binds reversibly to organelles, beads, or glass and generates a translocating force on a microtubule.     

Stable polymers of the axonal cytoskeleton: the axoplasmic ghost

We have examined the monomer-polymer equilibria which form the cytoskeletal polymers in squid axoplasm by extracting protein at low concentrations of monomer. The solution conditions inside the axon were matched as closely as possible by the extraction buffer (buffer P) to preserve the types of protein associations that occur in axoplasm. Upon extraction in buffer P, all of the neurofilament proteins in axoplasm remain polymerized as part of the stable neurofilament network. In contrast, most of the polymerized tubulin and actin in axoplasm is soluble although a fraction of these proteins also exists as a stable polymer. Thus, the axoplasmic cytoskeleton contains both stable polymers and soluble polymers. We propose that stable polymers, such as neurofilaments, conserve cytoskeletal organization because they tend to remain polymerized, whereas soluble polymers increase the plasticity of the cytoskeleton because they permit rapid and reversible changes in cytoskeletal organization.     

Cytoskeletal architecture and immunocytochemical localization of microtubule-associated proteins in regions of axons associated with rapid axonal transport: the beta,beta''-iminodipropionitrile-intoxicated axon as a model system

Axons from rats treated with the neurotoxic agent beta,beta'-iminodipropionitrile (IDPN) were examined by quick-freeze, deep-etch electron microscopy. Microtubules formed bundles in the central region of the axons, whereas neurofilaments were segregated to the periphery. Most membrane-bounded organelles, presumably including those involved in rapid axonal transport, were associated with the microtubule domain. The high resolution provided by quick-freeze, deep-etch electron microscopy revealed that the microtubules were coated with an extensive network of fine strands that served both to cross-link the microtubules and to interconnect them with the membrane-bounded organelles. The strands were decorated with granular materials and were irregular in dimension. They appeared either singly or as an extensive anastomosing network in fresh axons. The microtubule-associated strands were observed in fresh, saponin-extracted, or aldehyde-fixed tissue. To explore further the identity of the microtubule-associated strands, microtubules purified from brain tissue and containing the high molecular weight microtubule-associated proteins MAP 1 and MAP 2 were examined by quick-freeze, deep-etch electron microscopy. The purified microtubules were connected by a network of strands quite similar in appearance to those observed in the IDPN axons. Control microtubule preparations consisting only of tubulin and lacking the MAPs were devoid of associated strands. To learn which of the MAPs were present in the microtubule bundles in the axon, sections of axons from IDPN-treated rats were examined by immunofluorescence microscopy using antibodies to MAP 1A, MAP 1B, MAP 2, and tubulin. Anti-MAP 2 staining was only marginally detectable in the IDPN-treated axons, consistent with earlier observations. Anti-MAP 1A and anti-MAP 1B brightly stained the IDPN-treated axons, with the staining exclusively limited to the microtubule domains. Furthermore, thin section-immunoelectron microscopy using colloidal gold-labeled second antibodies revealed that both anti-MAP 1A and anti-MAP 1B stained fuzzy filamentous structures between microtubules. In view of earlier work indicating that rapid transport is associated with the microtubule domain in the IDPN-treated axon, it now appears that MAP 1A and MAP 1B may play a role in this process. We believe that MAP 1A and MAP 1B are major components of the microtubule-associated fibrillar matrix in the axon.     

THE ULTRASTRUCTURE OF A REPTILIAN MYONEURAL JUNCTION

Myoneural junctions in Anolis are characterized by the formation of troughs in the surface of the muscle fibers in which small branches of the terminal axon lie. The muscle surface membrane lining the troughs is thrown into complex branching and anastomosing folds, which compose the subneural apparatus of Couteaux. A compound membrane 500 to 700 A thick separates axoplasm from sarcoplasm at the endings. This consists of five distinct layers and is described in detail. A thin layer of cytoplasm (probably Schwann) separates terminal axoplasm from extracellular space at the surfaces of the junctional troughs. Terminal axoplasm lacks axoplasmic filaments and contains numerous vesicular or tubular appearing structures about 300 to 500 A in diameter. Both terminal axoplasm and sarcoplasm contain numerous mitochondria.     

Stable complexes of axoplasmic vesicles and microtubules: protein composition and ATPase activity

Fast transport of axonal vesicles and organelles is a microtubule-associated movement (Griffin, J. W., K. E. Fahnestock, L. Price, and P. N. Hoffman, 1983, J. Neuroscience, 3:557-566; Schnapp, B. J., R. D. Vale, M. P. Sheetz, and T. S. Reese, 1984, Cell, 40:455-462; Allen, R. D., D. G. Weiss, J. H. Hayden, D. T. Brown, H. Fujiwake, and M. Simpson, 1985, J. Cell Biol., 100:1736-1752). Proteins that mediate the interactions of axoplasmic vesicles and microtubules were studied using stable complexes of microtubules and vesicles (MtVC). These complexes formed spontaneously in vitro when taxol-stabilized microtubules were mixed with sonically disrupted axoplasm from the giant axon of the squid Loligo pealei. The isolated MtVCs contain a distinct subset of axoplasmic proteins, and are composed primarily of microtubules and attached membranous vesicles. The MtVC also contains nonmitochondrial ATPase activity. The binding of one high molecular mass polypeptide to the complex is significantly enhanced by ATP or adenyl imidodiphosphate. All of the axoplasmic proteins and ATPase activity that bind to microtubules are found in macromolecular complexes and appear to be vesicle-associated. These data allow the identification of several vesicle-associated proteins of the squid giant axon and suggest that one or more of these polypeptides mediates vesicle binding to microtubules.     

Microtrabecular structure of the axoplasmic matrix: visualization of cross-linking structures and their distribution

Axoplasmic transport is a dramatic example of cytoplasmic motility. Constituents of axoplasm migrate as far as 400 mm/d or at approximately 5 micron/s. Thin-section studies have identified the major morphological elements within the axoplasm as being microtubules, neurofilaments (100-A filaments), an interconnected and elongated varicose component of smooth endoplasmic reticulum (SER), more dilated and vesicular organelles resembling portions of SER, multivesicular bodies, mitochondria, and, finally, a matrix of ground substance in which the tubules, filaments, and vesicles are suspended. In the ordinary thin-section image, the ground substance is comprised of wispy fragments which, in not being noticeably tied together, do not give the impression of representing more than a condensation of what might be a homogeneous solution of proteins. With the high-voltage microscope on thick (0.5-micron) sections, we have noticed, however, that the so- called wispy fragments are part of a three-dimensional lattice. We contend that this lattice is not an artifact of aldehyde fixation, and our contention is supported by its visability after rapid-freezing and freeze-substitution. This lattice or microtrabecular matrix of axoplasm was found to consist of an organized system of cross-bridges between microtubules, neurofilaments, cisternae of the SER, and the plasma membrane. We propose that formation and deformation of this system are involved in rapid axonal transport. To facilitate electron microscope visualization of the trabecular connections between elements of axoplasm, the following three techniques were used: first, the addition of tannic acid to the primary fixative, OsO4 postfixation, then en bloc staining in uranyl acetate for conventional transmission electron microscope (TEM); second, embedding tissue in polyethylene glycol for thin sectioning, dissolving out the embedding medium from the sections and blocks, critical-point-drying (J. J. Wolosewick, 1980, J. Cell Biol., 86:675-681.), and then observing the matrix-free sections with TEM or the blocks with a scanning electron microscope; and third, rapid freezing of fixed tissue followed by freeze-etching and rotary- shadowing with replicas observed by TEM. All of these procedures yielded images of cross-linking elements between neurofilaments and organelles of the axoplasm. These improvements in visualization should enable us to examine the distribution of trabecular links on motile axonal organelles.     

NF-M is an essential target for the myelin-directed "outside-in" signaling cascade that mediates radial axonal growth

Neurofilaments are essential for acquisition of normal axonal calibers. Several lines of evidence have suggested that neurofilament-dependent structuring of axoplasm arises through an "outside-in" signaling cascade originating from myelinating cells. Implicated as targets in this cascade are the highly phosphorylated KSP domains of neurofilament subunits NF-H and NF-M. These are nearly stoichiometrically phosphorylated in myelinated internodes where radial axonal growth takes place, but not in the smaller, unmyelinated nodes. Gene replacement has now been used to produce mice expressing normal levels of the three neurofilament subunits, but which are deleted in the known phosphorylation sites within either NF-M or within both NF-M and NF-H. This has revealed that the tail domain of NF-M, with seven KSP motifs, is an essential target for the myelination-dependent outside-in signaling cascade that determines axonal caliber and conduction velocity of motor axons.     

A novel action of collapsin: Collapsin-1 increases antero- and retrograde axoplasmic transport independently of growth cone collapse

Chick collapsin-1, a member of the semaphorin family, has been implicated in axonal pathfinding as a repulsive guidance cue. Collapsin-1 induces growth cone collapse via a pathway which may include CRMP-62 and heterotrimeric G proteins. CRMP-62 protein is related to UNC-33, a nematode neuronal protein required for appropriately directed axonal extension. Mutations in unc-33 affect neural microtubules, the basic cytoskeletal elements for axoplasmic transport. Using computer-assisted video-enhanced differential interference contrast microscopy, we now demonstrate that collapsin-1 potently promotes axoplasmic transport. Collapsin-1 doubles the number of antero- and retrograde-transported organelles but not their velocity. Collapsin-1 decreases the number of stationary organelles, suggesting that the fraction of time during which a particle is moving is increased. Collapsin-1-stimulated transport occurs by a mechanism distinct from that causing growth cone collapse. Pertussis toxin (PTX) but not its B oligomer blocks collapsin-induced growth cone collapse. The holotoxin does not affect collapsin-stimulated axoplasmic transport. Mastoparan and a myelin protein NI-35 induce PTX-sensitive growth cone collapse but do not stimulate axoplasmic transport. These results provide evidence that collapsin has a unique property to activate axonal vesicular transport systems. There are at least two distinct pathways through which collapsin exerts its actions in developing neurons. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 316–328, 1997