The fibril forming proteins, tubulin and actin, play important roles in forming the cytoskeleton and in maintaining the architecture of the cell. Interactions of the cytoskeleton occur with the plasma membrane which affects the lateral mobility of the membrane proteins and also with organelles and processes which are crucial to such functions as mitosis, movement and motility, secretion, exonal transport and the mitogenic action of lectins. The importance of a complete understanding of the cytoskeleton and the range of interactions which are possible is further indicated by the demonstration that the cytoskeleton appears to be different in normal and tumor or transformed cells. Most, if not all, these functions necessitate some type of direct or indirect attachment of the cytoskeleton to some other cell component. Additional proteins other than tubulin and actin may be required for this attachment. We plan to determine the quantity of tubulin and actin in different types of membranes where there is reason to believe that interaction with the cytoskeleton exists in vivo and to determine the extent of tubulin and actin interactions with other proteins within the membrane. If it is possible to demonstrate growth of microtubules and microfilaments from membranes or granules in vitro, we will attempt to isolate and characterize the organizing centers and initiation sites. The effect of microtubule associated proteins and actin-binding proteins on initiation and the mechanism of growth of the fibrils will be examined. We plan to investigate the regulation and control of the initiation process and to relate it to changes in membrane structure which may be important, such as those brought about by the action of lectins. The myxomycete, Physarum polycephalum, will be used to study some of the developmental aspects of the mechanism by which microtubules attach to the membrane surrounding the nucleus. This organism has the advantage of having a large number of nuclei which are all in synchrony and contained within a single cell. Dark field microscopy will allow observation of the growth of single microtubules in real time.