Polylysine cross-links axoplasmic neurofilaments into tight bundles

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 2Ck313-3241. 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 (“shortchain”) polylysine did not inhibit the expansion of axoplasm and did not cause the axoplasm to condense. Inhibition of the expansion of axoplasm by long-chain pol ylysine was dependent on the polylysine concentration; condensation of axoplasm was observed at concentrations of 0.01 mg/ml (0.27 pM) 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 9lnm-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 crosslinked 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. 0 1995 Wiley-Liss, Inc.