BIOCHEMICAL INVESTIGATION OF NEUROFILAMENTS

点击翻译按钮获取中文摘要

The long-range goal of the proposed research is to understand, at the molecular level, the role of neurofilaments in the organization of the cytoskeletal network of mammalian neurons, and to contribute to understanding of the functioning of this network in the dynamics of axonal transport. The aim of the studies for the next three years is to relate the structure and biochemistry of neurofilaments to the structure and biochemistry of an associated complex that forms in vitro between neurofilaments and microtubules. Four interrelated approaches will be taken. (1) An ultracentrifugal, electron microscopic, and electrophoretic investigation will be carried out of the dissociation of neurofilaments into their component protofilaments. The intent is to define the protein composition and mass per unit length of the protofilaments as a first step in determining their substructure and mode of self-assembly. (2) Patterns of phosphorylation of the two large neurofilament proteins will be characterized by protein chemical methods to find out whether most of the phosphates are located in a small region of the proteins. The possible role of phosphorylation in stabilizing the structure of the neurofilaments will be investigated by enzymically perturbing the extent of phosphorylation, then assessing changes in the structure by ultracentrifugation and electron microscopy. Identification, by use of a covalently binding substrate analog, of the kinase responsible for the phosphorylation will be completed. (3) Investigation of the newly discovered microtubule-neurofilament associated complex will be continued. By chromatographic separation of the microtubule-associated proteins that mediate the complex and by viscometric assay of complex-formation, the specific protein(s) that act as cross-linkers will be identified, and the role of phosphorylation in determining their activity will be determined. By observation of light scattering from polystyrene beads incorporated into it, the internal mobility of the complex and the geometry of its internal spaces will be assessed. Attempts will be made to form the complex under conditions that will induce a higher degree of anisotropy in the complex than has so far been obtained. (4) A preliminary study will be carried out by light-scattering and viscometry to find out if actin filaments form an associated complex with neurofilaments.