Polylysine cross‐links axoplasmic neurofilaments into tight bundles

Abstract

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.

Mechanical Effects of Neurofilament Cross-bridges

“…Biological Relevance of in Vitro NF Gelation-The present study demonstrates that NF gels formed in vitro are more resistant to stress than any other noncovalent gel of biological polymers previously studied (34). However, NF in vivo are thought to be weakly cross-linked (47). The differences in these results and the modulation of NF rheology in vitro by agents such as acid phosphatase, PIP 2 , and F-actin support the hypothesis that NF cross-bridging is a NF-specific property that is strongly controlled in situ.…”

Visualization of single neurofilaments by immunofluorescence microscopy of splayed axonal cytoskeletons

“…For example, statistical analysis of neurofilament organization in axonal cross-sections has demonstrated that these polymers adopt a random distribution in regions where they are not packed densely, indicating that they do not have a strong affinity for each other [Price et al, 1993;Price et al, 1988]. Furthermore, studies on squid axons have shown that neurofilaments appear to move apart freely after axoplasm is separated from the axonal plasma membrane by extrusion [Brown & Lasek, 1993;Brown & Lasek, 1995]. Thus the tendency of neurofilaments to splay apart from each other in detergent-treated axons of cultured neurons is consistent with a growing body of evidence indicating that these cytoskeletal polymers are not extensively cross-linked to each other in axons.…”