Single Molecule Studies of Myosin-Ib

Myosin-Is are the single-headed, membrane-associated members of the myosin superfamily that are found in many eukaryotic cells. These actin-based motors have been shown to play important roles in powering membrane dynamics, defining cytoskeletal structure, and regulating mechanical signal-transduction. However, many molecular details of myosin-I function are not known. My goal has been to determine the mechanical and kinetic properties of a myosin-I isoform (myo1b) as it undergoes its force-generating power stroke under physiological tension and when external mechanical loads are applied to it. We therefore characterized the force dependence of myo1b splice isoforms using an optical trap and a novel isometric force clamp. Myo1b is alternatively spliced within the regulatory domain of the molecule, yielding motors that have “lever-arms” with different lengths. We found the actin-attachment kinetics of all myo1b splice isoforms to be highly force sensitive, with forces of \u3c 2 pN decreasing the rate of actin detachment \u3e 75 fold. However, we found that the magnitude of the tension sensitivities depend on the splice isoform. Therefore, we propose that the tension sensing properties of myo1b are transcriptionally regulated. Finally, we found the tension sensitivity of myo1b to be regulated by calcium, such that micromolar calcium concentrations effectively uncouple the myosin active site from lever arm rotation. Taken together, this work supports a model in which myosin-Is play roles in generating and sustaining membrane tension, and that the mechanochemical properties of this protein are regulated by alternative splicing and calcium