SPECTRIN-LIKE PROTEIN IN BRAIN

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We have completed all of the aims of the original proposal, as well as discovering brain protein 4.1, demonstrating the identity of brain 4.1 as synapsin I, and beginning the study of brain spectrin expression in mouse neuroblastoma cells and primary cultures of dorsal root ganglion neurons. In this application we describe experiments which are designed to give us a better understanding of brain spectrin structure, function, location, and interaction with synapsin I. The aims of this proposal are (1) to determine the domain structure of brain spectrin by combining peptide mapping with monoclonal antibody technology; (2) Quantitate the number of phosphate groups on the 235k Dal Beta subunit of brain spectrin. Localize these phosphates within the brain spectrin domain structure, and determine the function of the phosphate groups; (3) Quantitate the amount of the two brain spectrin subtypes within mouse brain with a quantitative immunodot assay; (4) Determine the precise location of brain spectrin subtypes, within specific neurons and glial cells by immunoelectronmicroscopy utilizing monoclonal antibodies; (5) Determine the domain structure of synapsin I, localizing the spectrin and synaptic vesicle binding domains. Quantitate the affinity and stoichiometry of the brain spectrin-synapsin I interaction and determine whether this interaction is regulated by synapsin I phosphorylation. Study the role of synapsin I in the brain spectrin-actin interaction; (6) Study the rate and stoichiometry of spectrin subunit synthesis and assembly within cultured S20Y mouse neuroblastoma cells and dorsal root ganglion (DRG) primary neuronal cultures; (7) Study the function of brain spectrin by microinjection of monoclonal antibodies which recognize only the brain spectrin (240/235) or brain spectrin (240/235E) subtypes, into neuroblastoma or primary DRG neurons. Observe the effect of spectrin antibody microinjection upon neuronal shape and neurite extension. Determine the effect upon microtubule, neurofilament, and actin microfilament organization.