The ultimate goal of this application is to investigate the basic mechanism underlying the assembly of neurofilaments and interactions between neurofilaments and other cytoplasmic organelles. We will explore a possible role of phosphorylation in such interactions. Assembly of neurofilaments will be monitored by sedimentation of purified, iodinated subunits. Phosphorylated forms of neurofilament proteins will be prepared by high efficiency liquid chromatography. Dephosphorylated forms of these proteins will be obtained by enzymatic treatment. Phosphate metabolism in neurofilaments will be explored by in vivo labeling techniques, 2D gel electrophoresis and scintillation. Developmental expression of neurofilament proteins will be studied by intraocular injection of radioactive amino acids, supplemented with morphometric analysis of filaments in the optic nerve. These studies will provide basic biochemical data which are not only relevant to our understanding of the structure, development and functions of neuronal filaments, but also by implication applicable to pathological conditions in which abnormality of filaments are observed, such as Alzheimer's and idiopathic Parkinson's disease.
