Motor domain-dependent localization of myo1b (myr-1)

Myosin-I is the single-headed, membrane binding member of the myosin superf amily that plays a role in membrane dynamics and transport [1-6]. Its molec ular functions and its mechanism of regulation are not known. In mammalian cells, myosin-I is excluded from specific microfilament populations, indica ting that its localization is tightly regulated. Identifying the mechanism of this localization, and the specific actin populations with which myosin- I interacts, is crucial to understanding the molecular functions of this mo tor. eGFP chimeras of myo1b [7] were imaged in live and fixed NRK cells. Ra tio-imaging microscopy shows that myo1b-eGFP concentrates within dynamic ar eas of the actin cytoskeleton, most notably in membrane ruffles. Myo1 b-eGF P does not associate with stable actin bundles or stress fibers. Truncation mutants consisting of the motor or tail domains show a partially overlappi ng cytoplasmic localization with full-length myo1b, but do not concentrate in membrane ruffles. A chimera consisting of the light chain and tail domai ns of myo1b and the motor domain from nonmuscle myosin-IIb (nmMIIb) concent rates on actin filaments in ruffles as well as to stress fibers. In vitro m otility assays show that the exclusion of myo1 b from certain actin filamen t populations is due to the regulation of the actomyosin interaction by tro pomyosin. Therefore, we conclude that tropomyosin and spatially regulated a ctin polymerization play important roles in regulating the function and loc alization of myo1b.