Abstract
Recently, extensive researches have reported that intracellular contractile forces play pivotal roles in cell functions, such as morphogenesis, differentiation, and homeostasis. Actin stress fibers are the main contractile force generator in non-muscle cells. Actin stress fibers have a less-organized sarcomeric structure constituted of actin, myosin, α-actinin, and other contraction-associated proteins. Their localization and activity of cytoskeletal molecules are highly regulated in a spatiotemporal manner. Moreover, dynamics and contractile properties of actin stress fibers are thought to be regulated by physical cues, for example, the tension externally applied to the actin stress fibers and internally generated by actomyosin contraction. To shed light on kinetics of actin stress fibers under various actomyosin activities, here we investigate the dynamics of fluorescent protein-tagged myosin molecules by fluorescence recovery after photobleaching (FRAP). We investigate the turnover rates and fractional recovery of myosin molecules associated with phosphomimic and nonphosphorylatable myosin regulatory light chain mutants (collaborative work with Dr. Masayuki Takahashi, Hokkaido Univ.). The double phosphomimic myosin regulatory light chain mutant (DD-mutant) had low fractional recovery. It indicates that myosin molecules with DD-mutant might have the longer lifetime attached to F-actin.
