Organization of mammalian neurofilament polypeptides within the neuronal cytoskeleton.

Abstract

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...

Nestin expression in repopulating mesangial cells promotes their proliferation

“…27 Nestin is furthermore suggested to act as a linker or cross-bridge protein between intermediate filaments, microfilaments and microtubules. 42,43 In rapidly dividing and migrating cells assembly and disassembly of filaments need to be strictly regulated. In a neuronal stem cell line, nestin was found to be reorganized during mitosis in.…”

Regional modulation of neurofilament organization by myelination in normal axons

“…In large neurons neurofilaments are the most abundant cytoskeletal organelles, and have been postulated to act as important intrinsic determinants of axonal caliber (Hoffman et al, 1987). Neurofilaments in vertebrates are composed of three related proteins with apparent molecular weights of 68, 150, 200 kDa (NF-L, NF-M, and NF-H, respectively) (Hoffman and Lasek, 1975;Liem et al, 1978;Geisler et al, 1983;Willard and Simon, 1983;Hirokawa et al, 1984;Schlaepfer, 1987). All three ofthese proteins share a central rod domain, but they differ in their C-terminal regions.…”

“…In NF-H, this region contains over 40 lysine-serine-proline repeats (Julien et al, 1986(Julien et al, , 1988Lees et al, 1988), which provide potential phosphorylation sites (Julien et al, 1983;Geisler et al, 1983;Lee et al, 1988). The extent of phosphorylation of this region, which contributes to the sidearms of the neurofilament (Hirokawa et al, 1984;Carden et al, 1985;Hisanaga and Hirokawa, 1988) appears to differ within different regions of the neuron, with a higher phosphorylation state in the axon than in the cell body and dendrite (Stemberger and Sternberger, 1983;Lee et al, 1986Lee et al, , 1987. One attractive hypothesis is that phosphorylation on the side arms may increase spacings between nearest-neighbor neurofilaments , and thereby achieve a larger axonal caliber.…”

Are cross-bridging structures involved in the bundle formation of intermediate filaments and the decrease in locomotion that accompany cell aging?

“…Perhaps the most well-defined IF-associated protein is the 200,000-D protein of the neurofilament triad. Evidence from both biochemical and immunocytochemical results suggests the carboxyl terminal of p200 as the possible cross-bridging structure that connects neurofilaments laterally in nerve axons (7,9,14). A similar candidate for such function is filaggrin, which functions as the bundling protein for keratin polypeptides in terminally differentiated keratinocytes (5).…”