This proposal is directed toward elucidating the mechanisms which regulate the spatial and temporal changes in actin filament organization that accompany growth cone movement during neuronal pathfinding. Actin filaments, the core structure of filopodia, are the major cytoskeletal component in nerve terminals. In order to understand actin filament dynamics in growth cones we must know: 1) in what form the actin arrives at the growing tip of the neuron; 2) where the actin monomer is incorporated into the filament; and 3,) what factors regulate its assembly and organization. We will utilize cultured neurons in an active state of growth to address these questions. Classical pulse-chase experiments followed by differential extraction to separate the soluble actin pool from he cytoskeletal actin will be combined with video imaging and fluorescence energy transfer experiments to examine the forms of actin and the region with the neuron where the actin assembles. Studies on the regulation of actin assembly and organization will focus on two proteins already demonstrated in other cell types to be of significance in actin assembly and membrane association. One of these proteins, actin depolymerizing factor (ADF), is regulated in other cells by phosphorylation and we have identified a phosphorylated form of ADF in growth cone particles. Since ADF abundant in neurons (20% to 40% of the level of actin on a molar basis), and since ADF binds actin with high affinity (Kd=0.1 uM), the amount and location of actin available for assembly will depend directly the nature of the membrane signal transduction mechanisms involved. A second protein, alpha-actinin, identified in neuronal growth cones, is a alpha-actinin in growth cone adhesion. Defined surface adhesion and cell adhesion molecules covalently attached to glass will be used to form adhesion sites on growth cones. The adhesion plague-associated molecules will be removed attached to the glass and the cytoplasmic proteins will be studies by immunogold electron microscopic methods. The data obtained will help us to understand how encounters between the growth cone and substratum affect the membrane attachment of actin and translate the environmental cues into an organized growth response.
