MECHANICAL EFFECTS OF NEUROFILAMENT CROSS-BRIDGES - MODULATION BY PHOSPHORYLATION, LIPIDS, AND INTERACTIONS WITH F-ACTIN

The structure of gels formed by bovine spinal cord neurofilaments was determined by fluorescence and electron microscopy and compared to mec hanical properties measured by their elastic and viscous response to s hear forces. Neurofilaments formed gels of high elastic modulus (>100 Pa) after addition of millimolar Mg2+. Gelation caused a slow increase in shear moduli to levels similar to those of vimentin intermediate f ilament networks, followed by a rapid rise due to formation of links b etween neurofilaments, mediated by cross-bridging structures that vime ntin filaments lack. Neurofilament gels are more resistant to large de formations than are vimentin networks, suggesting the importance of cr oss-bridges for neurofilament mechanical properties. Fluorescence imag ing of single neurofilaments showed flexible filaments that became str aighter when they adhered to glass or were incorporated into filament bundles. Electron microscopy of neurofilament gels showed a system of bundles intertwined within a more isotropic network of individual fila ments. Neurofilament gel formation was stimulated in vitro by acid pho sphatase treatment or by inositol phospholipids. In contrast, addition of actin filaments reduced the resistance of neurofilament gels to la rge stresses. These results suggest that dynamic and regulated interac tions occur between neurofilaments to form viscoelastic networks with properties distinct from other cytoskeletal structures.