Mark Willard

Expression of the growth-associated protein GAP-43 in adult rat retinal ganglion cells following axon injury

We have studied the expression of the growth-associated protein GAP-43 after injury to the axons of adult rat retinal ganglion cells (CNS neurons that do not normally regenerate injured axons). Both the biosynthetic labeling of GAP-43 and the GAP-43 immunoreactivity of the retina increased after axotomy, but only when the injury was within 3 mm of the eye. These results suggest the following conclusions: First, axon injury is sufficient to alter GAP-43 expression in CNS neurons, even in the absence of regeneration. Second, mechanisms that regulate GAP-43 expression are sensitive to the length of uninterrupted axon remaining after injury. Finally, the conditions that favor increased GAP-43 are similar to those that favor regrowth of injured CNS axons into grafts of peripheral nerve, suggesting that GAP-43 induction is accompanied by an increased potential of injured CNS neurons to regenerate.

Location of a protein of the fodrin-spectrin-TW260/240 family in the mouse intestinal brush border.

We have determined that a protein of the fodrin-spectrin-TW260/240 (FST) family is a component of the thin fibrils (approximately 5 nm wide, 100-200 nm long) that cross-link bundles of actin filaments to adjacent actin bundles and to the plasma membrane in the terminal web of the brush border of the intestinal epithelium. When isolated brush borders were incubated with anti-fodrin antibodies and prepared for electron microscopy by the quick-freeze, deep-etch technique, these approximately 5 nm fibrils were specifically decorated with the antibody. In addition, these cross-linking fibrils disappeared when the anti-fodrin-reactive proteins were extracted from the brush border. We conclude that FST is a component of a cross-linking system composed of approximately 5 nm fibrils that are morphologically distinct from the approximately 8 nm myosin-containing fibrils which were identified by anti-myosin decoration. In addition to linking actin bundles to adjacent actin bundles and to the plasma membrane, these FST fibrils may mediate actin-vesicle, actin-intermediate filament and vesicle-plasma membrane linkages.

Localization of fodrin during fertilization and early development of sea urchins and mice

Fodrin, a spectrin-like protein, is localized in gametes, zygotes, and embryos from sea urchins and mice. Mammalian fodrin comprises two polypeptides with molecular weights of approximately 240 kDa (alpha) and 235 kDa (beta). An antibody specific for mammalian alpha-fodrin cross-reacted with a 240-kDa polypeptide from sea urchin egg extracts. This indicates that sea urchins contain a protein of similar electrophoretic mobility and immunological properties to mammalian alpha-fodrin. When this antibody was used to stain the sea urchin gametes with indirect immunofluorescence, fodrin-specific fluorescence was localized to the acrosome of the sperm and was distributed over the entire egg near the surface in a punctate pattern similar to the distribution of polymeric actin. During sperm incorporation, the fodrin-specific fluorescence is found at the site of sperm incorporation, in the fertilization cone. After fertilization, the intensity of fodrin fluorescence increases. During mitosis and cytokinesis in sea urchins, the entire surface of the egg remains stained; the cleavage furrow also was stained but no more intensely than was the rest of the egg surface. Antibody labeling with colloidal gold followed by electron microscopy showed that fodrin was loated in the cytoplasm immediately beneath the plasma membrane. In unfertilized mouse oocytes, both actin and fodrin were stained most intensely beneath the membrane adjacent to the meiotic spindle. After insemination, the cell surfaces of the pronucleate egg and the second polar body were stained; however, the actin matrix surrounding the apposed pronuclei did not bind the fodrin antibody. During cytokinesis in the mouse, the cleavage furrow stained more intensely than did the rest of the egg cortex, and in embryos the cell borders were delineated. These results indicate that organisms as unrelated to mammals as sea urchins have fodrin-like proteins; the rearrangements of such proteins suggest that they participate in the actin-mediated events at the cell surface during fertilization and early development in both mice and sea urchins.

The composition and organization of axonally transported proteins in the retinal ganglion cells of the guinea pig.

We labeled the proteins of guinea pig retinal ganglion cells with [35S]methionine and analyzed the axonally transported polypeptides by means of sodium dodecyl sulfate gel electrophoresis. Five groups of transported polypeptides could be distinguished by their characteristic times of initial appearance in segments of the axons of the retinal ganglion cells. The times of initial appearance of the groups corresponded to maximum transport velocities ranging from greater than 200 mm/day to 0.5 mm/day. We directly compared these transported polypeptides to polypeptides undergoing axonal transport in the retinal ganglion cells of the rabbit. Electrophoretically similar polypeptides were transported at the same relative velocities in the two animals. Our results lead to the following conclusions. (1) The basic composition and organization of axonally transported proteins is probably a general constant feature of mammalian retinal ganglion cells, implying that the correct organization is important for the proper functioning of these neurons. Therefore, the results obtained by the analysis of individual model systems should have general significance. (2) Four discontinuities in the transport process (in addition to the 5 discontinuities represented by the major transport groups) were revealed by a consideration of subtle differences between the rabbit and guinea pig, as well as differences in the rate of disappearance of label from individual polypeptides within each transport group. (3) The guinea pig should provide a useful model system for studying axonal transport, especially for immunological studies, since antibodies against axonally transported proteins of the guinea pig can be conveniently prepared in the rabbit. (4) While the structure (as reflected by electrophoretic mobility) of most major axonally transported polypeptides appears to be conserved over the evolutionary period (about 30 million years) separating two orders of mammals, the electrophoretic mobility of one neurofilament-associated polypeptide, H, was abnormally variant between the two species.

The intra-axonal transport of polypeptide H: Evidence for a fifth (very slow) group of transported proteins in the retinal ganglion cells of the rabbit

We have determined that a genetically polymorphic polypeptide (H, molecular weight approximately equal to 195,000) of the rabbit nervous system is transported down the retinal ganglion cell axons at a velocity of 0.7-1.1 mm/day. The H-polypeptide and probably at least two additional polypeptides (molecular weights approximately 145,000 and 73,000) therefore compose a group of intra-axonally transported proteins which moves more slowly than the 4 groups previously described in these neurons. The polypeptides of this fifth group are similar in molecular weight to certain polypeptides transported slowly in other mammalian neurons.

GAPs and fodrin: Novel axonally transported proteins

Abstract The GAPs (growth associated proteins, axonally transported at elevated levels during periods of axon elongation) and fodrin (polypeptides associated with the outer cytoplasm of neurons and other cells) are novel, rapidly axonally transported proteins. The behavior of the GAPS suggests that the modulation of neuronal gene expression is a critical feature in regulating axon growth during development and regeneration. The properties of fodrin raise the possibility of a relationship between the process of axonal transport and other intracellular transport phenomena.

Electrophoretic Analysis of Axonally Transported Proteins in Toad Retinal Ganglion Cells

Abstract: As a preliminary step to studying changes in axonal transport in regenerating neurons, we have analyzed the composition and organization of polypeptides normally axonally transported in a neuronal system capable of regeneration, i.e., the retinal ganglion cells of the toad, Bufo marinus. We labeled proteins synthesized in the retina with 35S‐methionine and subsequently used one‐dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis to analyze labeled, transported proteins in tissues containing segments of the axons (the optic nerve, optic tract, and optic tecta) of the retinal ganglion cells. The transported polypeptides could be divided into five groups according to their apparent transport velocities. Many of the polypeptides of each group were electrophoretically similar to polypeptides of corresponding groups previously described in rabbit and guinea pig retinal ganglion cells, and in some cases, additional properties of the polypeptides indicated that the transported materials of the two vertebrate classes were homologous. These results serve two purposes. First they establish the retinal ganglion cells of the toad Bufo marinus as a model system in which changes in gene expression related to regeneration may be studied. Second they show that the organization and many aspects of the composition of axonal transport in retinal ganglion cells have been conserved in animals as unrelated as amphibians and mammals.

A GAP-43-like protein in cat visual cortex.

We have purified a protein that changes in relative concentration during the development of the kitten visual cortex. It resembles GAP-43 (a neuronal protein that is expressed at elevated levels during periods of development and regenerative axon growth) in the following respects: (1) it is an acidic protein (pI = 4.7) whose electrophoretic mobility on SDS-PAGE is similar to, but lower than rat GAP-43, suggesting that the cat protein is larger; (2) its electrophoretic mobility varies with the acrylamide concentration in a manner that is characteristic of GAP-43; (3) its concentration in kitten forebrain is elevated during early postnatal development; (4) the sequence of ten consecutive amino acids from a chemically generated fragment matches the expected sequence from GAP-43; and (5) its amino-acid content also matches GAP-43. We conclude that our purified protein is cat GAP-43. Immunoblots with an antibody prepared against rat GAP-43 suggested that the concentration of GAP-43 in the visual cortex declines with age.

Translocations of fodrin and its binding proteins

Fodrin, a protein related to erythrocyte spectrin, redistributes within the cell in certain situations. We compare such movements of fodrin and several fodrin binding proteins during the processes of axonal transport in neurons, and capping of surface proteins in lymphocytes. In neurons, three different populations of newly synthesized fodrin appear to be transported down the axons at different velocities corresponding to those of groups of transported proteins designated II, IV, and V. Actin, which can interact with fodrin, is transported at the velocity of group IV. Synapsin, a component of synaptic vesicles, is also reported to bind to fodrin. One population of synapsin is transported more rapidly than fodrin, at the velocity of group I: two additional populations of transported synapsin may overlap fodrin in groups II and IV. We consider possible functional associations of these different populations of fodrin and fodrin binding proteins. We note that the transport of group IV proteins resembles in certain respects the process of capping in lymphocytes, suggesting the possibility of a common mechanism. We outline one of several possible mechanisms.

Regulation of Axon Growth and Cytoskeletal Development

The mature, steady-state axon is a temporally ephemeral collection of matter whose apparent constancy reflects a delicate balance between the continuous introduction of materials from the cell body by the process of axonal transport and their exit. At a given instant, the major morphological manifestations of this steady state are the arrays of neurofilaments, microtubules, a variety of membrane bounded organelles, and the plasma membrane; these elements communicate with each other by a complex lattice of crosslinkers.