Neurofilaments in the axons of mammalian spinal cord neurons are extensively cross-linked ; consequently, the filaments and their cross-bridges compose a three-dimensional lattice . We have used antibody decoration in situ combined with tissue preparation by the quick-freeze, deep-etch technique to locate three neurofilament polypeptides (195, 145, and 73 Kd) within this lattice . When antibodies against each polypeptide were incubated with detergent-extracted, formaldehyde-fixed samples of rabbit spinal cord, each antibody assumed a characteristic distribution : anti-73-Kd decorated the neurofilament core uniformly, but not the cross-bridges ; anti-145-Kd also decorated the core, but less uniformly ; sometimes the anti-145-Kd antibodies were located over the bases of cross-bridges . In contrast, anti-195-Kd primarily decorated the cross-bridges between the neurofilaments . These observations show that the 73-Kd polypeptide is a component of the central core of neurofilaments, and that the 195-Kd polypeptide is a component of the inter-neurofilamentous cross-bridges . It is consistent with this conclusion that we found few cross-bridges between neurofilaments in the optic nerves of neonatal rabbits during a developmental period when the ratio of 195 to 73 or 145-Kd polypeptides is much lower than in adults . The ratio of 195-Kd polypeptide to the other two neurofilament polypeptides also appeared much lower in the cell bodies and dendrites than in axons of adult spinal cord neurons, when the dispositions of the three polypeptides were studied by immunofluorescence experiments. The cell bodies apparently contain neurofilaments composed primarily of 145-and 73-Kd polypeptides, because we observed antibody decoration of individual neurofilaments in the cell bodies with anti-73-and -145-Kd, but not with anti-195-Kd . We conclude that the 195-Kd polypeptide participates in a crosslinking function, and that this function is, at least in certain neurons, most prevalent in the mature axon .The axonal cytoskeleton is typically composed of longitudinally oriented neurofilaments and microtubules that are extensively cross-linked to themselves, to each other, and to the plasma membrane by thin fibrils (3,10,11,22,23,28,35,40,41). Embedded in the resulting three-dimensional lattice are membrane-bounded organelles (e .g., mitochondria, vesiculo-tubular structures, smooth endoplasmic reticulum) which are themselves extensively cross-linked to the neurofilaments, microtubules, or both. The system of cross-bridges appears to determine the spatial relationships between the various elements ofthe axoplasm ; in addition, the cross-links between elements that may be moving past each other by the process of axonal transport raise the question ofthe dynamic THE JOURNAL OF CELL BIOLOGY " VOLUME 98 APRIL 1984 1523-1536 0 The Rockefeller University Press -0021-9525/84/04/1523/14 $1 .00 properties of these cross-bridges (i.e., how they are made and released) and whether they could affect the translocation of organelles down the a...

Fodrin (formerly designated 26 and 27) comprises two polypeptides (250,000 and 240,000 mol wt) that are axonally transported at a maximum time-averaged velocity of 40 mm/d-slower than the most rapidly moving axonally transported proteins, but faster than at least three additional groups of proteins . In this communication, we report the intracellular distribution of fodrin . Fodrin was purified from guinea pig brain, and a specific antifodrin antibody was produced in rabbit and used to localize fodrin in tissue sections and cultured cells by means of indirect immunofluorescence . Fodrin antigens were highly concentrated in the cortical cytoplasm of neurons and also nonneuronal tissues (e .g., skeletal muscle, uterus, intestinal epithelium) . Their disposition resembles a lining of the cell: hence, the designation fodrin (from Greek fodros, lining) . In cultured fibroblasts, immunofluorescently labeled fodrin antigens were arranged in parallel arrays of bands in the plane of the plasma membrane, possibly reflecting an exclusion of labeled fodrin from some areas occupied by stress fibers. The distribution of fodrin antigens in mouse 3T3 cells transformed with simian virus 40 was more diffuse, indicating that the disposition of fodrin is responsive to altered physiological states of the cell. When mixtures of fodrin and F-actin were centrifuged, fodrin cosedimented with the actin, indicating that these proteins interact in vitro.We conclude that fodrin is a specific component of the cortical cytoplasm of many cells and consider the possibilities : (a) that fodrin may be indirectly attached to the plasma membrane via cortical actin filaments ; (b) that fodrin may be mobile within the cortical cytoplasm and that, in axons, a cortical lining may be in constant motion relative to the internal cytoplasm ; and (c) that fodrin could serve to link other proteins and organelles to a submembrane forcegenerating system .A multitude of proteins synthesized in the cell bodies of neurons are conveyed by the process of axonal transport to the axons and synaptic terminals, which are themselves unable to synthesize most proteins. In the retinal ganglion cells (the centrally projecting neurons of the retina) of mammals and amphibians, the axonally transported proteins can be roughly divided into five groups according to their apparent maximum transport velocities (18,24,38,45,46): the major proteins of the first group (transport velocity > 240 mm/d) are associated with membranous organelles resembling the plasma membrane (26); the second group (velocity = 30-60 mm/d) is heterogeneous and includes mitochondrial proteins and proteins asso-

Growth-associated protein, GAP-43, is a polypeptide that is induced in neurons when they grow axons. We show by means of subcellular fractionation and immunohistochemical localization that GAP-43 is a component of neuronal growth cones as well as growing neurites; it is similar to a major phosphoprotein, pp46, of a growth cone-enriched subcellular fraction. These conclusions are consistent with the possibility that the induction of GAP-43/pp46 is an important event in the establishment of a productive growth state in which a neuron is competent to extend an axon.

No abstract

In an effort to determine whether the "growth state" and the "mature state" of a neuron are differentiated by different programs of gene expression, we have compared the rapidly transported (group I) proteins in growing and nongrowing axons in rabbits . We observed two polypeptides (GAP-23 and GAP-43) which were of particular interest because of their apparent association with axon growth . GAP-43 was rapidly transported in the central nervous system (CNS) (retinal ganglion cell) axons of neonatal animals, but its relative amount declined precipitously with subsequent development. It could not be reinduced by axotomy of the adult optic nerves, which do not regenerate ; however, it was induced after axotomy of an adult peripheral nervous system nerve (the hypoglossal nerve, which does regenerate) which transported only very low levels of GAP-43 before axotomy. The second polypeptide, GAP-23 followed the same pattern of growth-associated transport, except that it was transported at significant levels in uninjured adult hypoglossal nerves and not further induced by axotomy. These observations are consistent with the "GAP hypothesis" that the neuronal growth state can be defined as an altered program of gene expression exemplified in part by the expression of GAP genes whose products are involved in critical growth-specific functions. When interpreted in terms of the GAP hypothesis, they lead to the following conclusions: (a) the growth state can be subdivided into a "synaptogenic state" characterized by the transport of GAP-23 but not GAP-43, and an "axon elongation state" requiring both GAPS; (b) with respect to the expression of GAP genes, regeneration involves a recapitulation of a neonatal state of the neuron ; and (c) the failure of mammalian CNS neurons to express the GAP genes may underly the failure of CNS axons to regenerate after axon injury .Certain rapidly transported proteins (which we have designated "growth-associated polypeptides" [GAPs]) are specifically induced during the regeneration of amphibian retinal ganglion cell axons (17). In the preceding paper we proposed that the GAPs are proteins involved in key functions related to axon growth and that their induction is a critical event in axon regeneration. The mammalian nervous system provides a particularly interesting and important system in which to further evaluate this hypothesis, because it is possible to distinguish between metabolic changes resulting from axon injury itself and those directly related to axon growth : mature neurons of the mammalian peripheral nervous system (PNS) (e.g., the hypoglossal and vagus nerves) retain the ability to regenerate their axons after injury, while those of the central nervous system (CNS) (e.g., the optic nerve and spinal cord) generally do not . The failure of CNS regeneration is an important clinical problem and in the past has been explained in terms of models emphasizing either extrinsic or intrinsic factors . A particularly durable extrinsic model has been that regenerating CNS axons are ph...