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R J Lasek

Publications and source records attributed to R J Lasek.

At least 73 records · Page 4Linked to original sources
PubMed1984

Axonal tubulin and axonal microtubules: biochemical evidence for cold stability.

Nerve extracts containing tubulin labeled by axonal transport were analyzed by electrophoresis and differential extraction. We found that a substantial fraction of the tubulin in the axons of the retinal ganglion cell of guinea pigs is not solubilized by conventional methods for preparation of microtubules from whole brain. In two-dimensional polyacrylamide gel electrophoresis this cold-insoluble tubulin was biochemically distinct from tubulin obtained from whole brain microtubules prepared by cold cycling. Cleveland peptide maps also indicated some differences between the cold-extractable and cold-insoluble tubulins. The demonstration of cold-insoluble tubulin that is specifically axonal in origin permits consideration of the physiological role of cold-insoluble tubulin in a specific cellular structure. It appears likely that much of this material is in the form of cold-stable microtubules. We propose that the physiological role of cold-insoluble tubulin in the axon may be associated with the regulation of the axonal microtubule complexes in neurons.

Animals↗
PubMed1984

A conditioning lesion of the peripheral axons of dorsal root ganglion cells accelerates regeneration of only their peripheral axons.

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 also exhibits accelerated outgrowth in response to a prior peripheral axotomy. In contrast to this, the fastest growing central branch axons of DRG cells, which normally regenerate at a rate of 2.5 mm/day, are not significantly affected by a prior peripheral axotomy. A population of more slowly growing axons in the dorsal root also does not exhibit accelerated outgrowth in response to a peripheral conditioning lesion. The results of these experiments indicate that changes in the DRG neuron's metabolism induced by prior axotomy of its peripheral axon do not affect the regenerative properties of both axons equally. This raises the possibility that accelerated axonal outgrowth in only one axonal branch results from a differentially regulated supply of proteins to the two axons by the DRG cell body.

Animals↗
PubMed1984

Regional differences in the neuronal cytoskeleton.

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 proteins, designated NF60 and NF65 , is 5 times more abundant in the axon than in the cell body. The relative amounts of actin and tubulin are comparable in these two regions of the neuron. In addition, the ratio of NF60 and NF65 is different in the cell body and axon. The cell bodies contain proportionally more NF60 than the axons. However, the physical properties of the tubulin in the cell body, as measured by relative solubility, differ from that of the axon. The substantial differences between the composition of the cytoskeleton of the cell bodies and axons of Aplysia suggests that at least two distinct cytoskeletal networks exist in these neurons, one specific for the cell body and the other specific for the axon.

Animals↗
PubMed1983

Bodian's silver method reveals molecular variation in the evolution of neurofilament proteins.

The recent demonstration that Bodian's silver method specifically stains mammalian neurofilament subunits (NFs), but not other intermediate filament proteins (IFs), provides a specific marker for the identification of neurofilament polypeptides. We have applied the Bodian stain to SDS-PAGE separated polypeptides in nervous tissues from 9 species, representing neuronal evolution in 4 major phyla: chordata, mollusca, arthropoda and annelida. Every species tested except the arthropod showed intense silver staining of a set of polypeptides, each subsequently identified as NFs by immunomethods. These results demonstrate that the affinity of NFs for Bodian's silver stain is conserved during the evolution of nervous systems in a diverse spectrum of animals. Further, considerable variation in the molecular weight of NF subunits was found among the 6 vertebrates studied. This variation suggests that the molecular weight of NFs has not been conserved during evolution, a quality which appears to be unusual for a structural protein.

Animals↗
PubMed1983

Neurofilamentous network and filamentous matrix preserved and isolated by different techniques from squid giant axon.

The contribution of the neurofilamentous network to the structure of the squid giant axon was analyzed electron-microscopically. Axial 10-nm filaments cross-linked by radial 5-nm bridges form a network that is present in preparations prepared by a variety of techniques. The axoplasm is differentiated into dense and less dense regions. In the presence of Co(II) ions, the neurofilamentous network was remarkably well preserved and appeared to be associated with a dense web of fine filament matrix, which also was identified in extracted axoplasm and in fractions enriched with neurofilament protein complex. In the presence of La(III) ions, the neurofilamentous network had a coarse and open appearance. The stereo images of extracted and critical-point dried axoplasm suggested that the neurofilamentous network contains ordered lattice-like regions. Extracted preparations of extruded axoplasm and fractions enriched with neurofilament protein complex suggested that the properties of the network are determined by the neurofilament protein complex. It is proposed that the neurofilamentous network is the essential determinant of the form of the axon, and that the order within the network is determined by the radial components of the network. The structures observed in the different preparations are not artifacts, but rather are related closely to their native state in the axon.

Animals↗
PubMed1983

Ontophyletics of the nervous system: development of the corpus callosum and evolution of axon tracts.

The evolution of nervous systems has included significant changes in the axon tracts of the central nervous system. These evolutionary changes required changes in axonal growth in embryos. During development, many axons reach their targets by following guidance cues that are organized as pathways in the embryonic substrate, and the overall pattern of the major axon tracts in the adult can be traced back to the fundamental pattern of such substrate pathways. Embryological and comparative anatomical studies suggest that most axon tracts, such as the anterior commissure, have evolved by the modified use of preexisting substrate pathways. On the other hand, recent developmental studies suggest that a few entirely new substrate pathways have arisen during evolution; these apparently provided opportunities for the formation of completely new axon tracts. The corpus callosum, which is found only in placental mammals, may be such a truly new axon tract. We propose that the evolution of the corpus callosum is founded on the emergence of a new preaxonal substrate pathway, the "glial sling," which bridges the two halves of the embryonic forebrain only in placental mammals.

Animals↗
PubMed1983

Correlation of axonal regeneration and slow component B in two branches of a single axon.

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 +/- 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 differences in regeneration rate. Slow axonal transport is classified into two distinct components: slow component a (SCa), corresponding to the microtubule/neurofilament network of the axonal cytoskeleton, and slow component b (SCb), corresponding to the microfilament complex/axoplasmic matrix. The transport rate of SCa and SCb in the peripheral sensory axons is approximately 2 times faster than their counterparts in the central sensory axons. SCa moves at 1.0 to 3.0 mm/day in the peripheral processes and 0.5 to 1.0 mm/day in the central processes; SCb moves at 3.5 to 6.5 mm/day in the peripheral processes and 2.0 to 3.5 mm/day in the central processes. In each branch of the DRG cell, the rate of axonal regeneration is similar to the rate of SCb transport. These results support the hypothesis that SCb is a rate-limiting factor in axonal regeneration because of its role in providing the cytoskeletal elements which are directly involved in the motility of the growth cone and elongation of the axon.

Animals↗
PubMed1983

Slowing of the axonal transport of neurofilament proteins during development.

We examined age-dependent changes in neurofilament transport in motor axons of the rat sciatic nerve. SDS-PAGE and gel fluorography confirmed that the distribution of labeled neurofilament triplet protein coincides with the major slow component a (SCa) wave in these neurons. The velocity of neurofilament transport was calculated on the basis of the location of the 50th percentile Of radioactivity in this wave 33 days after motor neurons were labeled by the intraspinal administration of [3H]leucine and [3H]lysine. Overall, the velocity fell from 1.95 mm/day at 3 weeks of age to 1.12 mm/day at 20 weeks. Between 3 and 10 weeks, it fell at a 6-fold higher rate (0.096 mm/day/week) than between 10 and 20 weeks (0.016 mm/day/week). We also found a marked change in the shape of the slow component wave during development. It appeared to consist of several overlapping peaks moving at slightly different velocities in animals 10 weeks of age or less as compared to a single slower moving peak at 20 weeks. We propose that the velocity of slow axonal transport reflects the level of maturation of the neuron, and that the presence of several overlapping peaks Of transported radioactivity in the sciatic nerve of younger animals reflects the presence of several populations of motor axons at different stages of development. We also discuss the relationship between changes in the velocity of neurofilament transport and alterations in the composition of the cytoskeleton that occur as the axon grows in caliber during postnatal development.

Animals↗
PubMed1982

Fast axonal transport in extruded axoplasm from squid giant axon.

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.

Adenosine Triphosphate↗
PubMed1982

Neurofilament protein phosphorylation. Species generality and reaction characteristics.

We have previously demonstrated that neurofilament protein (NFP) is endogenously phosphorylated in squid giant fiber axoplasm (1). Here the generality of this phenomenon is demonstrated, and optimal reaction conditions are described. Phosphorylation of Myxicola NFP is shown in vivo and in isolated axoplasm by 32P labeling from [32P]orthophosphoric acid and [gamma-32P]ATP, respectively. Phosphorylation of guinea pig NFP is shown in extracts of peripheral nerve and in enriched NFP preparations. Both Myxicola NFP subunits (150 and 160 kilodaltons) and all three guinea pig NFP subunits (200, 145, and 68 kilodaltons) are phosphorylated. Phosphorylation of 10-nm filament protein from cultured fibroblasts is also shown optimal reaction conditions with guinea pig enriched NFP are as follows. The reaction has high specificity for ATP as substrate. Divalent cations are required, Mn2+ being most effective. Monovalent cations decrease activity. The pH optimum is 8.0. Cyclic AMP and other low molecular weight factors have no significant modulatory effect.

Animals↗
PubMed1982

Development of the marginal zone in the rhombenecephalon of Xenopus laevis.

The marginal zone is the most superficial component of the developing central nervous system. In this study we describe the pattern of formation of the marginal zone in Xenopus laevis as seen with the light microscope and correlate its appearance with developing axon bundles seen with the electron microscope. The marginal zone was first identified at stage 26 on the ventrolateral aspects of the neural tube in restrictive foci throughout the medulla. By stage 28 this presumptive fiber area had expanded to occupy a longitudinal zone along the ventrolateral aspect of the rhombencephalon. The marginal zone continued expanding rostrocaudally, mediolaterally, and dorsoventrally coming to occupy a substantial area along the periphery of the central nervous system by the time of hatching (stage 36). Electron microscopic (EM) observations allowed us to identify axons and growth cones on the lateral borders of the neural tube at stage 22. Foci of marginal zones identified in the caudal medulla at stage 24 consisted of several fascicles grouped together on the ventrolateral surface. These fascicles, separated and surrounded by ependymal processes contained axons and growth cones. The pattern of marginal zone development reflected the addition of axons within fascicles and also the addition of new fascicles of axons.

Animals↗
PubMed1982

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.

Actins↗
PubMed1982

MG2+ or Ca2+-activated ATPase in squid giant fiber axoplasm.

A divalent cation-activated ATPase in axoplasm from the squid giant axon is described. The enzyme requires Mg2+ or Ca2+, has a K+ optimum of 60 mM, and has a pH optimum of 7.5. Several nucleotide triphosphates other than ATP can serve as substrates. The enzyme is inhibited by excess ATP or Mg2+. The enzyme is enriched in a rapidly sedimenting fraction of the axoplasm, and is eluted in the exclusion volume of a Sepharose 4B column, suggesting that it is associated with a highly aggregated structure. Comparison of the properties of enzyme with those of myosin and Na+-K+-ATPase suggests that differs from both of these enzymes. The enzyme has many similarities to vertebrate nerve ATPases previously described. The demonstration of the presence of this ATPase in squid axoplasm proves the neuronal localization of the enzyme.

Adenosine Triphosphatases↗