9405928 Axelrod When a biological cell undergoes a change of shape in either a submicroscopic region or over the whole cell, the intricate connections between cytoskeletal proteins and the plasma membrane undergo a reorganization. As a step toward elucidating the details of these processes, this project studies the rates of some of the rapid and reversible chemical events that occur in the submembrane region. Three subprojects focus on different but related aspects of this reversible chemistry. The first subproject measures the rates of reversible binding and unbinding among certain key cytoskeletal proteins and between these proteins and the membrane lipids. Surface diffusion rates of these proteins while they are nonspecifically adsorbed at the membrane is also measured and evaluated for its possible role in enhancing the reaction rate to specific sites. The particular proteins whose interactions are to be examined are all actin- binding ones; myosin I, the spectrin/calmodulin system, protein 4.1, and actin itself. The second subproject examines the rapid time course changes in local calcium ion in the immediate submembrane region, in contrast to the transient changes farther into the cell, that act as a trigger for actin polymerization in some cases and exocytosis of secretory vesicles in other cases. The third project examines the rate of cholesterol transmembrane translocation (flip-flop) which is believed to affect the cell surface's plasticity during membrane shape changes and may even play a role in regulating those changes. %%% This project examines the speed of reactions that occur just beneath the membrane of biological cells. These reactions are very likely to be important in determining the cells' mechanical properties, its ability to move around, and its response to external natural chemicals. Because these reactions are localized in a very thin region, this project also develops or extends novel optical techniques particularly useful for s tudying biochemistry at surfaces, techniques that should find applications beyond the particular experiments here. The novel optical techniques are based around modifications of fluorescence microscopes, so that binding and unbinding of molecules to membrane surfaces, random diffusion of molecules for small distances along these surfaces, rapid changes of salt concentrations right near surfaces, and flipping of molecules from one side of a membrane to the other, can be detected and the corresponding rates can be measured. There are three main subprojects. The first looks at the dynamics of formation and dissolution of protein filaments that form a biological cell's "skeleton" at the points where it connects with the cell surface from the inside. In order to interpret the results and draw some general conclusions, a variety of living and model biological systems will be studied. The second subproject examines whether the calcium ion concentration just beneath the membrane changes in a different manner than the calcium ion concentration farther inside the cell upon chemical stimulation of the cell. Calcium is believed to play a central role in the triggering of several membrane reactions, and this project offers a fluorescence method to examine the calcium chemistry in a highly localized region. The third subproject examines the rates at which lipid molecules flip from one side of the membrane to the other. This flipping can have important effects on the cells mechanical properties and the reactions that occur in the membrane. ***
