The experiments of this proposal are part of a long-term goal to define the cellular processes that regulate the organization of the neuronal cytoskeleton. These experiments focus on neurofilaments (NF) and neurofilament proteins (NFP). NF are prominent components of the axonal cytoskeleton which contribute to its volume, stability, and form, and thereby the radial dimension of axons. NFP are phosphorylated in vivo. It is generally assumed that phosphorylation regulates various properties of NF, although the nature of these properties and how phosphorylation regulates them are unknown. For example, little information is available concerning where in the neuron NFP phosphorylation occurs, whether NFP phosphorylation is coordinated with dephosphorylation, or when phosphorylation occurs relative to the synthesis and assembly of NFP. Experiments are proposed in this application to directly address all of these issues. These experiments will use well defined culture systems coupled with electrophoretic, immunoprecipitation, and immunoblotting assays of NFP to analyze NFP phosphorylation (and dephosphorylation) in the neuron as a whole and in preparations of cell bodies or pure axons. An important parameter of NF organization is the lateral spacing between NF. Experiments are also proposed to evaluate the contribution of the various NFP to the packing of NF and to test the hypothesis that phosphorylation of NFP regulates the spacing between NF. These experiments will make use of a recently published procedure for quantifying the effect of various factors on the spacing of linear polymers in vitro. NF of varying composition and phosphorylation state will be prepared from purified NFP or by enzymatic treatment of native NF. The NF will be pelleted by centrifugation, and the spacing of NF in the pellets will be determined directly by electron microscopy and also by measuring the volume of the pellet. Completion of the proposed experiments will clarify several issues concerning the regulation of NF organization, and thus will provide a cellular and molecular framework for understanding the regulation of neuronal geometry. In addition, many degenerative and toxic disorders of the nervous system are manifest at a cellular level in dramatic alterations in the NF network. By defining normal mechanisms for the control of NF organization, the proposed experiments will provide information that is essential for understanding the cellular bases of these disorders.
