The Axolemma—Ectoplasm Complex of Squid Giant Axon

Metuzals, J.; Clapin, D. F.; Tasaki, Ichiji

doi:10.1007/978-1-4615-9337-9_3

Cited by 10 publications

(5 citation statements)

References 46 publications

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“…Fine filamentous material was often seen to project at right angles to the neurofilament, specifically from the beadlike portions. These thinner filaments, -5 nm in diameter, are comparable to the crossbridge associations previously described in the squid Adelman, 1980, 1983;Rice et al, 1980;Metuzals et al, 1982Metuzals et al, , 1983a, as well as in other organisms (Ellisman and Porter, 1980 between parallel neurofilaments. Apparent cross-bridges also show beadlike portions, although they are somewhat smaller (-8-16 nm) than those on the main portion of the neurofilament (Fig.…”

Section: Western Blot Analysis and Dephosphorylation Studiessupporting

confidence: 83%

“…1) in squid is attached to isolated 10 nm filaments and may be involved in cross-bridge structure (Rice et al, 1980;Metuzals et al, 1982Metuzals et al, , 1983b. Metuzals et al (1982Metuzals et al ( , 1983a have tentatively assigned the 380 kDa (HMW) and 220 kDa polypeptides to the cross-linking, or radial, 5 nm filaments and the 60 kDa polypeptide to the axial, or 10 nm, filaments.…”

Section: Discussionmentioning

confidence: 99%

“…The greatest concentration is at the periphery, where they are arranged in bundles; here they are more parallel than in central regions (Metuzals and Izzard, 1969;Hodge and Adelman, 1980). A continuous 3-dimensional network in the giant axon, consisting of neurofilaments formed by intercoiled unit filaments with cross-bridge associations forming ladderlike patterns, has been described; squid neurofilaments also contain distinct "beadlike" portions along their length at regular intervals (Metuzals, 1969;Metuzals and Izzard, 1969;Adelman, 1980, 1983;Rice et al, 1980;Roslansky et al, 1980;Metuzals et al, 1981aMetuzals et al, , b, 1982Metuzals et al, , 1983aVillegas and Villegas, 1984).…”

Section: Monoclonalmentioning

confidence: 99%

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Biochemical and immunocytochemical characterization and distribution of phosphorylated and nonphosphorylated subunits of neurofilaments in squid giant axon and stellate ganglion

Rs¹,

Pant²,

House³

et al. 1987

J. Neurosci.

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Monoclonal antibodies to squid neurofilament (aNFP) and intermediate filament (aIFA) proteins were used as probes for the biochemical and immunocytochemical analyses of neurofilament structure and distribution in the squid giant axon and stellate ganglion. On Western blots the aNFP antibody stained exclusively the 220 kDa and high-molecular-weight (HMW) components of neurofilaments in the giant axon, whereas the aIFA antibody primarily labeled the 60 kDa protein in the giant axon and the 60 and 65 kDa proteins in the stellate ganglion. Dephosphorylation of axoplasmic proteins by alkaline phosphatase resulted in a decrease in the molecular weights of both the 220 kDa and HMW neurofilament proteins and a concomitant loss of reactivity with the aNFP antibody on Western blots. This indicated that the aNFP antibody is specific for a phosphorylated epitope in the neurofilament. Increased dephosphorylation of the 220 kDa protein led to an enhanced immunostaining of the resultant 190 kDa polypeptide by the aIFA antibody, suggesting that the phosphorylated epitope may mask the conserved epitope recognized by aIFA. Light and electron microscopic immunocytochemical studies show intense labeling by the aNFP antibody in the giant axon. In contrast, the aIFA antibody labeled the glial cells around the giant axon intensely, while labeling of the giant axon itself was considerably less than that with the aNFP antibody. Since the 60 kDa protein in axoplasm is intensely stained by the aIFA antibody on Western blots, the relatively low amounts of labeling seen on semithin and thin sections of the giant axon by this antibody may be due to the masking of the 60 kDa protein by in situ fixed axoplasmic proteins. However, the aIFA antibody intensely labeled glial cells within the stellate ganglion and "islands" of filaments and nuclear membranes within ganglion cells. No reactivity for either antibody was seen in synapses. The aNFP antibody specifically labeled "beadlike" portions and cross-bridges on the axonal neurofilaments, suggesting that these components consist of the 220 kDa and HMW proteins. In contrast, the aIFA antibody labeled relatively smooth filaments in ganglion and glial cells. These data suggest that the 65 kDa protein represents the squid glial filament protein and that the 60 kDa protein found in axoplasm represents the low-molecular-weight subunit in the axonal neurofilament. The latter appears to be formed and/or organized in "islands" of filaments within ganglion cells.(ABSTRACT TRUNCATED AT 400 WORDS)

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Section: Western Blot Analysis and Dephosphorylation Studiessupporting

confidence: 83%

Section: Discussionmentioning

confidence: 99%

Section: Monoclonalmentioning

confidence: 99%

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Biochemical and immunocytochemical characterization and distribution of phosphorylated and nonphosphorylated subunits of neurofilaments in squid giant axon and stellate ganglion

Rs¹,

Pant²,

House³

et al. 1987

J. Neurosci.

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“…These MT-containing bundles were most numerous at the extreme periphery of the axoplasm (within a few pm of the surface), where many bundles of three or more MTs were observed. This is consistent with electron microscopic observations of axoplasm in the intact squid giant axon which have shown that MTs are most numerous in the peripheral axoplasm that underlies the plasma membrane [Metuzals et al, 1983a;Sakai et al, 19851. These ultrastructural observations indicate that long-chain polylysine binds tightly to neighboring NFs in isolated axoplasm and cross-links them together into tightly packed bundles, thereby preventing the normal tendency for these polymers to disperse radially into the surrounding solution.…”

Section: Electron Microscopysupporting

confidence: 87%

Polylysine cross‐links axoplasmic neurofilaments into tight bundles

Brown

Lasek

1995

Cell Motil. Cytoskeleton

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We have used axoplasm from the squid giant axon to investigate the effects of anionic and cationic polypeptides on the mobility and organization of axonal neurofilaments (NFs). Intact cylinders of axoplasm were extruded from squid giant axons into an excess volume of artificial axoplasm solution. In a previous study on the mobility of NFs in extruded axoplasm, we showed that these polymers disperse freely and diffusively into the surrounding solution, thereby expanding the axoplasmic cross-sectional area [Brown and Lasek, 1993: Cell Motil. Cytoskeleton 26:313-324]. In the present study, we found that 83nm-long ("long-chain") polylysine, a synthetic multivalent cationic protein, inhibited the radial expansion of isolated axoplasm and condensed the axoplasm, thereby reducing the cross-sectional area. Equivalent concentrations of a 7nm-long ("short-chain") polylysine did not inhibit the expansion of axoplasm by long-chain polylysine was dependent on the polylysine concentration; condensation of axoplasm was observed at concentrations of 0.01 mg/ml (0.27 microM) or greater. Electron microscopy of the condensed axoplasm showed that the NFs were aligned side-by-side and in parallel in closely-packed bundles. Equivalent concentrations of 91 nm-long ("long-chain") polyglutamate, a synthetic multivalent anionic protein, partially inhibited the expansion of axoplasm but did not cause the NFs to bundle and did not cause the axoplasm to condense. These studies indicate that cationic proteins bind tightly to the highly charged anionic surfaces of NFs and can link them together into compact bundles in a charge-dependent and length-dependent manner. The tightly packed organization of these cross-linked NFs differs from the normal loose organization of NFs in healthy axons. However, tightly bundled NFs are sometimes found in certain neuropathologies, such as giant axonal neuropathy.

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“…This was unexpected because actin has been shown repeatedly to migrate more rapidly ahead of the wave of tubulin and neurofilaments and to localise subaxolemmally [Hoffman and Lasek, 1975;Mori et al, 1979;Metuzals and Distance from the ganglion Fig. 6.…”

Section: Discussionmentioning

confidence: 92%

Effects of taxol on slow and fast axonal transport

Komiya

Tashiro

1988

Cell Motil. Cytoskeleton

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Axonal transport of tubulin in the rat sciatic nerve is almost completely inhibited by a single subepineural injection of taxol, without affecting that of neurofilament proteins. Actin and a large number of polypeptides cotransported with actin as minor components are also blocked by taxol, although to a lesser extent. Fast axonal transport is essentially free from the inhibitory effect of this drug. Although previous models have suggested that slow axonal transport involves the bulk movement of cytoskeletal structures, these results suggest that such transport may involve an equilibrium between polymerised and depolymerised forms of the axonal cytoskeleton.

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The Axolemma—Ectoplasm Complex of Squid Giant Axon

Cited by 10 publications

References 46 publications

Biochemical and immunocytochemical characterization and distribution of phosphorylated and nonphosphorylated subunits of neurofilaments in squid giant axon and stellate ganglion

Biochemical and immunocytochemical characterization and distribution of phosphorylated and nonphosphorylated subunits of neurofilaments in squid giant axon and stellate ganglion

Polylysine cross‐links axoplasmic neurofilaments into tight bundles

Effects of taxol on slow and fast axonal transport

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