Eukaryotic cells can maintain their shape despite a myriad of environmental perturbants, yet locally alter it in response to appropriate stimuli to engage in phagocytosis or secretion, or develop a whole cell response allowing the cell to locomote. The normal execution of these functions is necessary for the proper functioning of the immune system, the endocrine system as well as the orderly renewal of cells in the body. Aberrations of these functions may be involved in the mechanism of cell injury and neoplasia. The peripheral cytoplasm or cortex of these cells is thought to be responsible for the execution of these cell functions. The main constituent of the cortex is a network composed of actin filaments crosslinked by either filamin a 540kD protein or alpha-actinin, a 220kD calcium sensitive protein, or both. In addition, gelsolin, a 93 kD protein is also present which is able to cleave actin filaments thereby solating the network in the presence of calcium. The change in shape responsible for cell movement involves the generation of a force which protrudes the cell. The ability of this force to create movement must conform to the ability of the cytoplasmic network to flow or may involve cycles of network formation and dissolution in response to stimulation. Numerous questions remain as to what role these proteins play in these complex cell functions. The purpose of this grant proposal is to determine a number of important physical properties of systems composed of these purified proteins in order to further define this role. In particular, the rheologic or flow properties of actin crosslinked by filamin or alpha-actinin, or both at various ratios and total protein concentrations will be measured. These measurements will be facilitated by a new device which allows the biochemical alteration of a preformed network in the measuring device without the need for mechanical mixing and disruption of the structure. This will allow the measurement of protein samples which have concentrations similar to that found in the intact cell and importantly, allow the study of the formation and dissolution of the network in a completely undisturbed way. In addition the osmotic properties of these systems will be studied, to determine the ability of this mechanism to generate force, and the changes in electrical properties which occur upon application of an osmotic stress will be determined to explore the possibility that there is electrical coupling between the membrane and the cytoskeleton. A determination of the structures and hydrodynamic properties of these systems will be made by performing microscopic quasi- elastic light scattering and microscopic fluorescent photobleaching recovery on similar samples which are placed in dialysis fibers.
