Structure and Assembly of Neurofilaments (NF): Analysis of Native NF and of Specific NF Subunit Combinations

Abstract

A large body of information on the primary and secondary structure of intermediate filaments (IF) is now available [Weber et al., 1983; Steinert et al., 1985]. In the quest to ultimately understand IF structure, function and interaction at the molecular level, next steps include: (i) establishing the rules by which IF polypetides assemble into supramolecular structures; (ii) determining the three-dimensional (3-D) molecular architecture of IF; and (iii) establishing correlations between the molecular structure and function of the various IF. Because it has not been possible to obtain crystalline arrays of IF proteins in a consistent manner - which is a prerequisite for X-ray diffraction analysis of proteins - investigators have sought alternative techniques to approach these questions. In our labs we have begun to apply in vitro reconstitution of filaments and scanning transmission electron microscopy (STEM) to the study of neurofilaments (NF). This is a preliminary report on our work with NF, including effective and reproducible separation of the three NF polypeptides, in vitro reconstitution of NF from individual as well as specific combinations of subunits, and STEM mass measurements of native and reconstituted NF. We have observed that in vitro reassembly of NF takes place within strict limits of temperature, pH and salt concentration. In addition, STEM mass measurements disclose a marked polymorphism both of native and reconstituted NF.KeywordsIntermediate FilamentScanning Transmission Electron MicroscopyFilamentous StructureIntermediate FilamentKeratin FilamentThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.