The hypothesis that glial cells synthesize proteins which are transferred to adjacent neurons was... more The hypothesis that glial cells synthesize proteins which are transferred to adjacent neurons was evaluated in the giant fiber of the squid (Loligo pealei). When giant fibers are separated from their neuron cell bodies and incubated in the presence of radioactive amino acids, labeled proteins appear in the glial cells and axoplasm. Labeled axonal proteins were detected by three methods: extrusion of the axoplasm from the giant fiber, autoradiography, and perfusion of the giant fiber. This protein synthesis is completely inhibited by puromycin but is not affected by chloramphenicol. The following evidence indicates that the labeled axonal proteins are not synthesized within the axon itself. (a) The axon does not contain a significant amount of ribosomes or ribosomal RNA. (b) Isolated axoplasm did not incorporate [(3)H]leucine into proteins. (c) Injection of Rnase into the giant axon did not reduce the appearance of newly synthesized proteins in the axoplasm of the giant fiber. These ...
The proteins of the three major rate components of axonal transport in guinea pig retinal ganglio... more The proteins of the three major rate components of axonal transport in guinea pig retinal ganglion cells were analyzed by one- and two-dimensional gel electrophoresis. Each rate component consisted of a different set of proteins that remained associated with each other during transport. This suggests that each rate component represents a distinct macromolecular complex and that these complexes may be definable organelles such as microtubules, microfilaments, and smooth endoplasmic reticulum. Thus, the transport of radiolabeled proteins in the axon reflects the movement of complete subcellular rather than the movement of individual proteins.
The recent demonstration that Bodian's silver method specifically stains mammalian neurofila... more The recent demonstration that Bodian's silver method specifically stains mammalian neurofilament subunits (NFs), but not other intermediate filament proteins (I.
Axons from eyes transplanted to the tail in Xenopus larvae enter the caudal spinal cord and follo... more Axons from eyes transplanted to the tail in Xenopus larvae enter the caudal spinal cord and follow two adjacent tracts rostrally to the level of the cerebellum. When eyes are transplanted to the ear area, optic axons enter the hindbrain and follow the same tracts rostrally and caudally. These sensory pathways normally contain the embryonic sensory system of the Rohon-Beard axons and the descending and ascending tracts of nerve V. We propose that the transplanted optic axons have followed a continuous substrate sensory pathway normally shared by a number of different sensory tracts.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1984
Regional differences in the neuronal cytoskeleton were investigated in the giant neurons of Aplys... more Regional differences in the neuronal cytoskeleton were investigated in the giant neurons of Aplysia. Using SDS-PAGE, we have compared the proteins which comprise the cytoskeletons of cell bodies and axons. Separate populations of cell bodies and axons were collected and the proteins stained by the Coomassie brilliant blue method. Individual identified cell bodies, with long segments of their axons attached, were isolated, and the proteins were labeled with the [125I]Bolton-Hunter reagent. The proteins which are stably associated with the cytoskeleton were obtained by extracting the neuronal material in a physiological buffer containing Triton X-100. As a correlative measure to the biochemical analyses, electron microscopy was performed on the cell body and axonal fractions. Our results demonstrate that the composition and biochemical properties of the cytoskeletal proteins in the neuron cell bodies differ from those associated with axons. Specifically, the amount of neurofilament pr...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1984
Axotomy of the peripheral axon of dorsal root ganglion (DRG) cells is known to result in chromato... more Axotomy of the peripheral axon of dorsal root ganglion (DRG) cells is known to result in chromatolysis and changes in protein synthesis in DRG cells. We investigated whether a stimulus produced by peripheral branch axotomy would affect the regenerative properties of both the central and peripheral axon of the DRG cell equally. To examine this question, a conditioning crush lesion was made distally on the sciatic nerve 2 weeks prior to a testing lesion of either the dorsal root or peripheral branch axon near the DRG. Fast axonal transport of radioactive proteins was used to assess regeneration of DRG axons. In the adult rat, leading peripheral branch axons normally regenerate at a rate of 4.4 mm/day. If a conditioning lesion of the sciatic nerve is made 2 weeks before the test lesion, the rate of peripheral branch axonal regeneration increases by 25% to 5.5 mm/day. This effect is not limited to the fastest growing axons in the nerve since a population of more slowly growing axons als...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1985
Aplysia californica has been used to study the protein synthetic response of nervous tissue to st... more Aplysia californica has been used to study the protein synthetic response of nervous tissue to stress induced by elevated temperatures. The abdominal and pleural ganglia as well as associated connectives were exposed to various temperatures for 30 min, labeled with [33S]methionine at room temperature, and then analyzed by sodium dodecyl sulfate gel electrophoresis. All cells examined responded to temperatures of greater than 31 degrees C by a reduction in levels of labeled actin, as well as by the enhanced labeling of proteins with apparent Mr of 70,000 and 110,000. Two-dimensional electrophoresis indicated that the molecular weight and isoelectric focusing properties are similar to the heat shock proteins (HSPs) observed in other systems. In addition to these major HSPs, heat-induced proteins with molecular weights ranging from 70,000 to 90,000 were highly labeled in the neurosecretory bag cells. Further cell type-specific differences in the protein synthetic response to elevated t...
We investigated the relationship between slow axonal transport and axonal regeneration in the rat... more We investigated the relationship between slow axonal transport and axonal regeneration in the rat dorsal root ganglion (DRG) cell. The DRG cell sends out a single axon which bifurcates within the ganglion; one axon proceeds centrally into the spinal cord and the other proceeds peripherally. The rate of axonal regeneration is approximately 2 times faster for the peripheral processes (4.6 + 0.9 mm/day) than for the central processes (2.1 f 0.5 mm/day). The peripheral and central processes regenerate through dissimilar environments (sciatic nerve and dorsal root, respectively); thus, environmental factors may account for the differences in regeneration rates. We tested this possibility by measuring the regeneration of motoneuron axons within the ventral root (histologically similar to the dorsal root). The motoneuron regeneration rate within the ventral root is similar to the motoneuron regeneration rate within the sciatic nerve, suggesting that factors within the DRG cell produce the ...
Incubation of intracellulary perfused squid giant axons in [3H]leucine demonstrated that newly sy... more Incubation of intracellulary perfused squid giant axons in [3H]leucine demonstrated that newly synthesized proteins appeared in the perfusate after a 45-min lag period. The transfer of labeled proteins was shown to occur steadily over 8 h of incubation, in the presence of an intact axonal plasma membrane as evidenced by the ability of the perfused axon to conduct propagated action potentials over this time-period. Intracellularly perfused RNase did not affect this transfer, whereas extracellularly applied puromycin, which blocked de novo protein synthesis in the glial sheath, prevented the appearance of labeled proteins in the perfusate. The uptake of exogenous 14C-labeled bovine serum albumin (BSA) into the axon had entirely different kinetics than the endogenous glial labeled protein transfer process. The data provide support for the glia-neuron protein transfer hypothesis.
Neurofilaments were isolated from the axoplasm of the giant axons of Myxicola infundibulum and sq... more Neurofilaments were isolated from the axoplasm of the giant axons of Myxicola infundibulum and squid. The axoplasm was fractionated by discontinuous sucrose gradient centrifugation and gel filtration on Sepharose 4B. The fractions were monitored for neurofilaments by electron microscopy. When isolated in the presence of chelating agents, the neurofilaments of Myxicola are composed almost entirely of protein subunits with mol wt of 150,000 and 160,000. Squid neurofilaments contain two major proteins with mol wt of 200,000 and 60,000. These proteins are compared with other intermediate filament proteins which have been reported in the literature.
To assay the detailed structural relationship between axonally transported vesicles and their sub... more To assay the detailed structural relationship between axonally transported vesicles and their substrate microtubules, vesicle transport was focally cold blocked in axoplasm that was extruded from the squid giant axon. A brief localized cold block concentrated anterogradely and retrogradely transported vesicles selectively on either the proximal or or distal side of the block. Normal movement of the concentrated vesicles was reactivated by rewarming the cold-blocked axoplasm. At the periphery of the axoplasm, moving vesicles were located on individual microtubules that had become separated from the other cytomatrix components. The presence of moving vesicles on isolated microtubules permitted the identification of the structural components required for vesicle transport along microtubules. The results show that 16-18-nm cross-bridges connect both anterogradely and retrogradely moving vesicles to their substrate microtubules. These observations demonstrate that cross-bridges are funda...
The distribution and length of actin microfilaments (MF) was determined in axoplasm extruded from... more 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 axopla...
The distribution of the proteins migrating with the slow components a (SCa) and b (SCb) of axonal... more The distribution of the proteins migrating with the slow components a (SCa) and b (SCb) of axonal transport were studied in cross-sections of axons with electron microscope autoradiography. Radiolabeled amino acids were injected into the hypoglossal nucleus of rabbits and after 15 d, the animals were killed. Hypoglossal nerves were processed either for SDS-polyacrylamide gel electrophoresis fluorography to identify and locate the two components of slow transport, or for quantitative electron microscope autoradiography. Proteins transported in SCa were found to be uniformly distributed within the cross-section of the axon. Labeled SCb proteins were also found throughout the axonal cross-section, but the subaxolemmal region of the axon contained 2.5 times more SCb radioactivity than any comparable area in the remainder of the axon.
We have observed the phosphorylation of neurofilament protein from squid axoplasm. Phosphorylatio... more We have observed the phosphorylation of neurofilament protein from squid axoplasm. Phosphorylation is demonstrated by 32P labeling of protein during incubation of axoplasm with [gamma-32P]ATP. When the labeled proteins are separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two bands, at 2.0 x 10(5) daltons and greater than 4 x 10(5) daltons, contain the bulk of the 32P. The 2.0 x 10(5)-dalton phosphorylated polypeptide comigrates on SDS-PAGE with one of the subunits of squid neurofilament protein. Both major phosphorylated polypeptides co-fractionate with neurofilaments in discontinuous sucrose gradient centrifugation and on gel filtration chromatography on Sepharose 4B. The protein-phosphate bond behaves like a phospho-ester, and labeled phospho-serine is identified in an acid hydrolysate of the protein. The generality of this phenomenon in various species and its possible physiological significance are discussed.
The hypothesis that transported vesicles are preferentially associated with a subclass of microtu... more The hypothesis that transported vesicles are preferentially associated with a subclass of microtubules has been tested in lobster axons. A cold block was used to collect moving vesicles in these axons; this treatment caused the vesicles to accumulate in files along some of the microtubules. Quantitative analysis of the number of vesicles associated with microtubule segments indicated that lobster axons have two distinct populations of microtubules--transport microtubules that are the preferred substrates for vesicle transport and architectural microtubules that contribute to axonal structure.
Regenerating sensory axons in the dorsal roots of adult mammals are stopped at the junction betwe... more Regenerating sensory axons in the dorsal roots of adult mammals are stopped at the junction between the root and spinal cord by reactive astrocytes. Do these cells stop axonal elongation by activating the physiological mechanisms that normally operate to stop axons during development, or do they physically obstruct the elongating axons? In order to distinguish these possibilities, the cytology of the axon tips of regenerating axons that were stopped by astrocytes was compared with the axon tips that were physically obstructed at a cul-de-sac produced by ligating a peripheral nerve. The terminals of the physically obstructed axon tips were distended with neurofilaments and other axonally transported structures that had accumulated when the axons stopped elongating. By contrast, neurofilaments did not accumulate in the tips of regenerating axons that were stopped by spinal cord astrocytes at the dorsal root transitional zone. These axo-glial terminals resembled the terminals that axons make on target neurons during normal development. On the basis of these observations, astrocytes appear to stop axons from regenerating in the mammalian spinal cord by activating the physiological stop pathway that is built into the axon and that normally operates when axons form stable terminals on target cells.
Development of video-enhanced contrast-differential interference contrast for light microscopy ha... more Development of video-enhanced contrast-differential interference contrast for light microscopy has permitted study of both orthograde and retrograde fast axonal transport of membranous organelles in the squid giant axon. This process was found to continue normally for hours after the axoplasm was extruded from the giant axon and removed from the confines of the axonal plasma membrane. It is now possible to follow the movements of the full range of membranous organelles (30-nanometer vesicles to 5000-nanometer mitochondria) in a preparation that lacks a plasma membrane or other permeability barrier. This observation demonstrates that the plasma membrane is not required for fast axonal transport and suggests that action potentials are not involved in the regulation of fast transport. Furthermore, the absence of a permeability barrier surrounding the axoplasm makes this an important model for biochemical pharmacological, and physical manipulations of membranous organelle transport.
Neurofilaments purified from invertebrate giant axons have been analyzed with the electron micros... more Neurofilaments purified from invertebrate giant axons have been analyzed with the electron microscope . The neurofilaments have a helical substructure which is most easily observed when the neurofilaments are partially denatured with 0.5 M KCl or 2 M urea . When the ropelike structure comprising the neurofilaments untwists, two strands 4-5.5nm in diameter can be resolved . Upon further dena- turation these strands break up into rod-shaped segments and subsequently these segments roll up into amorphous globular structures . Stained, filled densities can be resolved within the strand segments, and these resemble similar structures observed within the intact neurofilaments . The strands appear to consist of protofilaments 2-2.5 nm in diameter. These observations suggest that the neuro- filament is a ropelike, helical structure composed of two strands twisted tightly around each other, and they support the filamentous rather than the globular model of intermediate filament structure .
The hypothesis that glial cells synthesize proteins which are transferred to adjacent neurons was... more The hypothesis that glial cells synthesize proteins which are transferred to adjacent neurons was evaluated in the giant fiber of the squid (Loligo pealei). When giant fibers are separated from their neuron cell bodies and incubated in the presence of radioactive amino acids, labeled proteins appear in the glial cells and axoplasm. Labeled axonal proteins were detected by three methods: extrusion of the axoplasm from the giant fiber, autoradiography, and perfusion of the giant fiber. This protein synthesis is completely inhibited by puromycin but is not affected by chloramphenicol. The following evidence indicates that the labeled axonal proteins are not synthesized within the axon itself. (a) The axon does not contain a significant amount of ribosomes or ribosomal RNA. (b) Isolated axoplasm did not incorporate [(3)H]leucine into proteins. (c) Injection of Rnase into the giant axon did not reduce the appearance of newly synthesized proteins in the axoplasm of the giant fiber. These ...
The proteins of the three major rate components of axonal transport in guinea pig retinal ganglio... more The proteins of the three major rate components of axonal transport in guinea pig retinal ganglion cells were analyzed by one- and two-dimensional gel electrophoresis. Each rate component consisted of a different set of proteins that remained associated with each other during transport. This suggests that each rate component represents a distinct macromolecular complex and that these complexes may be definable organelles such as microtubules, microfilaments, and smooth endoplasmic reticulum. Thus, the transport of radiolabeled proteins in the axon reflects the movement of complete subcellular rather than the movement of individual proteins.
The recent demonstration that Bodian's silver method specifically stains mammalian neurofila... more The recent demonstration that Bodian's silver method specifically stains mammalian neurofilament subunits (NFs), but not other intermediate filament proteins (I.
Axons from eyes transplanted to the tail in Xenopus larvae enter the caudal spinal cord and follo... more Axons from eyes transplanted to the tail in Xenopus larvae enter the caudal spinal cord and follow two adjacent tracts rostrally to the level of the cerebellum. When eyes are transplanted to the ear area, optic axons enter the hindbrain and follow the same tracts rostrally and caudally. These sensory pathways normally contain the embryonic sensory system of the Rohon-Beard axons and the descending and ascending tracts of nerve V. We propose that the transplanted optic axons have followed a continuous substrate sensory pathway normally shared by a number of different sensory tracts.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1984
Regional differences in the neuronal cytoskeleton were investigated in the giant neurons of Aplys... more Regional differences in the neuronal cytoskeleton were investigated in the giant neurons of Aplysia. Using SDS-PAGE, we have compared the proteins which comprise the cytoskeletons of cell bodies and axons. Separate populations of cell bodies and axons were collected and the proteins stained by the Coomassie brilliant blue method. Individual identified cell bodies, with long segments of their axons attached, were isolated, and the proteins were labeled with the [125I]Bolton-Hunter reagent. The proteins which are stably associated with the cytoskeleton were obtained by extracting the neuronal material in a physiological buffer containing Triton X-100. As a correlative measure to the biochemical analyses, electron microscopy was performed on the cell body and axonal fractions. Our results demonstrate that the composition and biochemical properties of the cytoskeletal proteins in the neuron cell bodies differ from those associated with axons. Specifically, the amount of neurofilament pr...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1984
Axotomy of the peripheral axon of dorsal root ganglion (DRG) cells is known to result in chromato... more Axotomy of the peripheral axon of dorsal root ganglion (DRG) cells is known to result in chromatolysis and changes in protein synthesis in DRG cells. We investigated whether a stimulus produced by peripheral branch axotomy would affect the regenerative properties of both the central and peripheral axon of the DRG cell equally. To examine this question, a conditioning crush lesion was made distally on the sciatic nerve 2 weeks prior to a testing lesion of either the dorsal root or peripheral branch axon near the DRG. Fast axonal transport of radioactive proteins was used to assess regeneration of DRG axons. In the adult rat, leading peripheral branch axons normally regenerate at a rate of 4.4 mm/day. If a conditioning lesion of the sciatic nerve is made 2 weeks before the test lesion, the rate of peripheral branch axonal regeneration increases by 25% to 5.5 mm/day. This effect is not limited to the fastest growing axons in the nerve since a population of more slowly growing axons als...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 1985
Aplysia californica has been used to study the protein synthetic response of nervous tissue to st... more Aplysia californica has been used to study the protein synthetic response of nervous tissue to stress induced by elevated temperatures. The abdominal and pleural ganglia as well as associated connectives were exposed to various temperatures for 30 min, labeled with [33S]methionine at room temperature, and then analyzed by sodium dodecyl sulfate gel electrophoresis. All cells examined responded to temperatures of greater than 31 degrees C by a reduction in levels of labeled actin, as well as by the enhanced labeling of proteins with apparent Mr of 70,000 and 110,000. Two-dimensional electrophoresis indicated that the molecular weight and isoelectric focusing properties are similar to the heat shock proteins (HSPs) observed in other systems. In addition to these major HSPs, heat-induced proteins with molecular weights ranging from 70,000 to 90,000 were highly labeled in the neurosecretory bag cells. Further cell type-specific differences in the protein synthetic response to elevated t...
We investigated the relationship between slow axonal transport and axonal regeneration in the rat... more We investigated the relationship between slow axonal transport and axonal regeneration in the rat dorsal root ganglion (DRG) cell. The DRG cell sends out a single axon which bifurcates within the ganglion; one axon proceeds centrally into the spinal cord and the other proceeds peripherally. The rate of axonal regeneration is approximately 2 times faster for the peripheral processes (4.6 + 0.9 mm/day) than for the central processes (2.1 f 0.5 mm/day). The peripheral and central processes regenerate through dissimilar environments (sciatic nerve and dorsal root, respectively); thus, environmental factors may account for the differences in regeneration rates. We tested this possibility by measuring the regeneration of motoneuron axons within the ventral root (histologically similar to the dorsal root). The motoneuron regeneration rate within the ventral root is similar to the motoneuron regeneration rate within the sciatic nerve, suggesting that factors within the DRG cell produce the ...
Incubation of intracellulary perfused squid giant axons in [3H]leucine demonstrated that newly sy... more Incubation of intracellulary perfused squid giant axons in [3H]leucine demonstrated that newly synthesized proteins appeared in the perfusate after a 45-min lag period. The transfer of labeled proteins was shown to occur steadily over 8 h of incubation, in the presence of an intact axonal plasma membrane as evidenced by the ability of the perfused axon to conduct propagated action potentials over this time-period. Intracellularly perfused RNase did not affect this transfer, whereas extracellularly applied puromycin, which blocked de novo protein synthesis in the glial sheath, prevented the appearance of labeled proteins in the perfusate. The uptake of exogenous 14C-labeled bovine serum albumin (BSA) into the axon had entirely different kinetics than the endogenous glial labeled protein transfer process. The data provide support for the glia-neuron protein transfer hypothesis.
Neurofilaments were isolated from the axoplasm of the giant axons of Myxicola infundibulum and sq... more Neurofilaments were isolated from the axoplasm of the giant axons of Myxicola infundibulum and squid. The axoplasm was fractionated by discontinuous sucrose gradient centrifugation and gel filtration on Sepharose 4B. The fractions were monitored for neurofilaments by electron microscopy. When isolated in the presence of chelating agents, the neurofilaments of Myxicola are composed almost entirely of protein subunits with mol wt of 150,000 and 160,000. Squid neurofilaments contain two major proteins with mol wt of 200,000 and 60,000. These proteins are compared with other intermediate filament proteins which have been reported in the literature.
To assay the detailed structural relationship between axonally transported vesicles and their sub... more To assay the detailed structural relationship between axonally transported vesicles and their substrate microtubules, vesicle transport was focally cold blocked in axoplasm that was extruded from the squid giant axon. A brief localized cold block concentrated anterogradely and retrogradely transported vesicles selectively on either the proximal or or distal side of the block. Normal movement of the concentrated vesicles was reactivated by rewarming the cold-blocked axoplasm. At the periphery of the axoplasm, moving vesicles were located on individual microtubules that had become separated from the other cytomatrix components. The presence of moving vesicles on isolated microtubules permitted the identification of the structural components required for vesicle transport along microtubules. The results show that 16-18-nm cross-bridges connect both anterogradely and retrogradely moving vesicles to their substrate microtubules. These observations demonstrate that cross-bridges are funda...
The distribution and length of actin microfilaments (MF) was determined in axoplasm extruded from... more 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 axopla...
The distribution of the proteins migrating with the slow components a (SCa) and b (SCb) of axonal... more The distribution of the proteins migrating with the slow components a (SCa) and b (SCb) of axonal transport were studied in cross-sections of axons with electron microscope autoradiography. Radiolabeled amino acids were injected into the hypoglossal nucleus of rabbits and after 15 d, the animals were killed. Hypoglossal nerves were processed either for SDS-polyacrylamide gel electrophoresis fluorography to identify and locate the two components of slow transport, or for quantitative electron microscope autoradiography. Proteins transported in SCa were found to be uniformly distributed within the cross-section of the axon. Labeled SCb proteins were also found throughout the axonal cross-section, but the subaxolemmal region of the axon contained 2.5 times more SCb radioactivity than any comparable area in the remainder of the axon.
We have observed the phosphorylation of neurofilament protein from squid axoplasm. Phosphorylatio... more We have observed the phosphorylation of neurofilament protein from squid axoplasm. Phosphorylation is demonstrated by 32P labeling of protein during incubation of axoplasm with [gamma-32P]ATP. When the labeled proteins are separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two bands, at 2.0 x 10(5) daltons and greater than 4 x 10(5) daltons, contain the bulk of the 32P. The 2.0 x 10(5)-dalton phosphorylated polypeptide comigrates on SDS-PAGE with one of the subunits of squid neurofilament protein. Both major phosphorylated polypeptides co-fractionate with neurofilaments in discontinuous sucrose gradient centrifugation and on gel filtration chromatography on Sepharose 4B. The protein-phosphate bond behaves like a phospho-ester, and labeled phospho-serine is identified in an acid hydrolysate of the protein. The generality of this phenomenon in various species and its possible physiological significance are discussed.
The hypothesis that transported vesicles are preferentially associated with a subclass of microtu... more The hypothesis that transported vesicles are preferentially associated with a subclass of microtubules has been tested in lobster axons. A cold block was used to collect moving vesicles in these axons; this treatment caused the vesicles to accumulate in files along some of the microtubules. Quantitative analysis of the number of vesicles associated with microtubule segments indicated that lobster axons have two distinct populations of microtubules--transport microtubules that are the preferred substrates for vesicle transport and architectural microtubules that contribute to axonal structure.
Regenerating sensory axons in the dorsal roots of adult mammals are stopped at the junction betwe... more Regenerating sensory axons in the dorsal roots of adult mammals are stopped at the junction between the root and spinal cord by reactive astrocytes. Do these cells stop axonal elongation by activating the physiological mechanisms that normally operate to stop axons during development, or do they physically obstruct the elongating axons? In order to distinguish these possibilities, the cytology of the axon tips of regenerating axons that were stopped by astrocytes was compared with the axon tips that were physically obstructed at a cul-de-sac produced by ligating a peripheral nerve. The terminals of the physically obstructed axon tips were distended with neurofilaments and other axonally transported structures that had accumulated when the axons stopped elongating. By contrast, neurofilaments did not accumulate in the tips of regenerating axons that were stopped by spinal cord astrocytes at the dorsal root transitional zone. These axo-glial terminals resembled the terminals that axons make on target neurons during normal development. On the basis of these observations, astrocytes appear to stop axons from regenerating in the mammalian spinal cord by activating the physiological stop pathway that is built into the axon and that normally operates when axons form stable terminals on target cells.
Development of video-enhanced contrast-differential interference contrast for light microscopy ha... more Development of video-enhanced contrast-differential interference contrast for light microscopy has permitted study of both orthograde and retrograde fast axonal transport of membranous organelles in the squid giant axon. This process was found to continue normally for hours after the axoplasm was extruded from the giant axon and removed from the confines of the axonal plasma membrane. It is now possible to follow the movements of the full range of membranous organelles (30-nanometer vesicles to 5000-nanometer mitochondria) in a preparation that lacks a plasma membrane or other permeability barrier. This observation demonstrates that the plasma membrane is not required for fast axonal transport and suggests that action potentials are not involved in the regulation of fast transport. Furthermore, the absence of a permeability barrier surrounding the axoplasm makes this an important model for biochemical pharmacological, and physical manipulations of membranous organelle transport.
Neurofilaments purified from invertebrate giant axons have been analyzed with the electron micros... more Neurofilaments purified from invertebrate giant axons have been analyzed with the electron microscope . The neurofilaments have a helical substructure which is most easily observed when the neurofilaments are partially denatured with 0.5 M KCl or 2 M urea . When the ropelike structure comprising the neurofilaments untwists, two strands 4-5.5nm in diameter can be resolved . Upon further dena- turation these strands break up into rod-shaped segments and subsequently these segments roll up into amorphous globular structures . Stained, filled densities can be resolved within the strand segments, and these resemble similar structures observed within the intact neurofilaments . The strands appear to consist of protofilaments 2-2.5 nm in diameter. These observations suggest that the neuro- filament is a ropelike, helical structure composed of two strands twisted tightly around each other, and they support the filamentous rather than the globular model of intermediate filament structure .
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