Examination of the Kinetics of Myosin VI during Endocytosis

Abstract

The molecular motor myosin VI has been implicated in endocytosis, a trafficking pathway mediating intracellular uptake of items such as membrane receptors and nutrients. Previous studies demonstrate the localization of the large insert isoform of myosin VI to clathrin-coated vesicles and the no insert isoform of myosin VI to early endosomes. The kinetics of myosin VI functionality on these endocytic structures has not yet been examined, however. This study uses fluorescence recovery after photobleaching (FRAP) to examine the turnover kinetics of the no insert and large insert isoforms of myosin VI during endocytosis. The results demonstrate that myosin VI turns over dynamically on endocytic structures and that different isoforms on different intracellular compartments have distinct turnover rates. The FRAP assay system is then implemented with a novel live cell expression of an artificial dimer of myosin VI to demonstrate the dimeric functionality of myosin VI on endocytic structures in vivo. Further studies of the turnover rates of the myosin VI binding partner Dab2 on clathrin-coated structures demonstrate that Dab2 turns over more rapidly than full length myosin VI, a novel indication of the relative turnover rates of a motor protein versus its binding partner on a given cellular compartment. The turnover kinetics of specific myosin VI motor domain mutants on endocytic structures are also examined, e.g. the D179Y mutant implicated in progressive hearing loss, a study which offers insight into the functional source of myosin VI-related deafness. In addition to providing insight into the endocytic functionality of myosin VI, these FRAP studies offer general insight into the dynamics of myosin motor proteins on intracellular structures, the functional differences between isoforms of given myosin proteins, and the comparative turnover rates of myosin proteins and their binding partners.