In vitro polymorphism and phase transitions of the neurofilamentous network isolated from the giant axon of the squid (Loligo pealei L.).

Using electron microscopy (EM), optical diffraction and image reconstruction techniques, we have demonstrated polymorphism of neurofilamentous network (NFN) in vitro based on phase transitions of the protein assemblies. The specific polymorphic appearances depended upon a number of factors, such as K+, Mg2+, Ca2+ ions, as well as the charge and hydration state of the molecules. Furthermore, modifications initiated by the state of phosphorylation of the sidearm proteins played an important role, especially in determining the sidearm disposition of the NFN. The Ca2+-activated protease removed the sidearms. Other enzymes activated by Ca2+ may initiate new association patterns of the peptide remnants and the intercoiling of two smooth neurofilaments (NFs) into paired helical filament-like (PHF-like) strands. Prolonged storage of the isolated NFs in Rubinson-Baker solution resulted in autocrosslinking and intercoiling of modified NFN components. The in vitro polymorphism and phase transitions of squid NFN induced under controlled conditions have been compared to modifications of cytoskeleton observed by EM in frontal lobe biopsies of Alzheimer patients. We conclude that similar processes, as induced in vitro, do occur in neurons of Alzheimer patients.