R3 - Mechano-chemistry and regulation of myosin-I -E. Michael Ostap, Ph.D. Myosin-I is the single-headed membrane-associated member of the myosin superfamily that is found in mosteukaryotic cells. Recent investigations suggest that these motors play crucial roles in membrane dynamics, cytoskeletalstructure, mechanical signal transduction, and endosome processing. However, little is known about the force-generating and regulatory properties of myosin-I isoforms or about the mechanical properties of the myosin-I-membrane interaction. Therefore, we will utilize state-of-the-art biochemical and biophysical techniques to determinethe basic mechanical and regulatory properties ofmyosin-I isoforms. We will investigate the following specific aims:1, Regulation of myosin-I by calcium and calmodulin. We will determine the effects of calcium on myosinATPase activities, and we will investigate the role of calcium binding on calmodulin affinity and lever arm structure.We will also focus on understanding how the different myosin-I subclasses are regulated and on understanding howalternative splicing in the lever arm affects regulation.2. Mechanical properties of myosin-I isoforms. We propose that the different myosin-I subclasses are biochemicallyand mechanically tuned for performing different cell biological functions. We will test this proposal by investigatingmechanical and force generating properties of myosin subclasses. Biochemical and structural experiments indicate thatmyosin-I isoforms may act as force-sensors in a manner consistent with the Fenn Effect, i.e., strain on the myosinaffects biochemical and structural transitions altering the lifetimes of the strong-binding states. Therefore, we will alsodetermine the lifetimes of force-producing states during isometric tension for the different myosin isoforms. We willalso determine the effect of calcium regulation on the force generating and mechanical properties ofmyosin-I isoforms,and we will correlate these funding with the biochemical and structural results obtained in Aim l.3. Force generation by lipid-associated myosin-I. Myosin-I isoforms associate with cellular membranes viainteractions between the positively charged myosin-I tail domains and acidic phospholipids. Nothing is known aboutthe ability of myosin-I to generate force relative to a lipid membrane, and very little is known about the strength of themyosin-I-lipid connection. Therefore, we will determine strength of the myosin-lipid interaction, and we will measurethe ability of myosin to produce force while bound to fluid lipid bilayers.
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