Mechanical effects of neurofilament cross-bridges. Modulation by phosphorylation, lipids, and interactions with F-actin

Abstract

The structure of gels formed by bovine spinal cord neurofilaments was determined by fluorescence and electron microscopy and compared to mechanical properties measured by their elastic and viscous response to shear 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 filament networks, followed by a rapid rise due to formation of links between neurofilaments, mediated by cross-bridging structures that vimentin filaments lack. Neurofilament gels are more resistant to large deformations than are vimentin networks, suggesting the importance of cross-bridges for neurofilament mechanical properties. Fluorescence imaging of single neurofilaments showed flexible filaments that became straighter 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 filaments. Neurofilament gel formation was stimulated in vitro by acid phosphatase treatment or by inositol phospholipids. In contrast, addition of actin filaments reduced the resistance of neurofilament gels to large stresses. These results suggest that dynamic and regulated interactions occur between neurofilaments to form viscoelastic networks with properties distinct from other cytoskeletal structures.