Myosin V: We have shown by immunolocalization that myosin V is concentrated on melanosomes (MS), the specialized pigment-producing organelle of melanocytes (MC). A working model for melanosome movement within mammalian MCs was proposed in which the movement is microtubule- based centrally and actomyosin V-based peripherally (i.e. within MC dendrites and dendritic tips). The shape and pigment distribution of wild type MCs and MCs devoid of myosin V were compared both in vitro (using primary MC cultures) and in situ. In both cases, the principal defect in mutant MCs was found to be in the transport of MSs to the periphery of the cell rather than in cell shape, as was previously thought. Taken together, our studies indicate that the absence of a myosin V-dependent MS transport system, which functions to move MSs from the cell body to dendritic tips, is responsible for the coat color defect in myosin V- mice. Immunolocalizations of myosin V in nerve growth cones implicate this motor in organelle motility within neurons as well. Myosin XI: Dictyostelium myoJ, a class XI unconventional myosin, was shown by immunofluorescence microscopy to be concentrated on the membranes of the contractile vacuole (CV) complex. Analyses of myoJ- cells created by targeted gene disruption revealed, however, that myoJ is not required for normal CV functioning. MyoJ- cells are also normal as regards chemotactic aggregation and endocytosis. Biochemical studies indicate that myoJ uses calmodulin as a light chain. Myosin I: The light chain (LC) of Acanthamoeba myosin IC (AMIC) was cloned by RT-PCR and subsequent screening of a cDNA library. A full- length cDNA for the AMIC heavy chain was constructed and coexpressed with the LC as a fully-active enzyme in bacculovirus-infected cells. Several proteins that bind to the SH3 domain of Dictyostelium myosin IC have been identified. Myosin II: The flexibilities and bend angles of minifilaments assembled using wild-type and mutant Acanthamoeba myosin II rod domains were determined by electric birefringence. The results confirm that the flexibility of the rod resides entirely within the hinge domain, and indicate that while hinge residues other than the proline confer significant flexibility, the proline is required for the sharp bend to occur. The thermal unfolding of these molecules has also been studied. ER Dynamics: A chimeric GFP molecule was designed to ensure retention in the endoplasmic reticulum (ER)and used to show loss of ER membrane continuity within starfish eggs upon fertilization.
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