Myosin VIIa is an unconventional myosin widely expressed in organisms ranging from amoebae to mammals that has been shown to play vital roles in cell adhesion and phagocytosis. We have studied Drosophila myosin VIIa that was expressed in Sf9 cells. We have shown that this myosin has high duty ratio kinetics similar to that of processive motors, but we also showed that it did not easily dimerize even if the full length molecule was expressed in Sf9 cells. The enzymatic activity of full length Drosophila myosin VIIa is regulated by an intramolecular folding event. A binding partner (termed M7BP for myosin 7 binding partner) for FLM7a was identified using the C-terminal FERM domain of the myosin as a bait in a yeast two hybrid screen. The binding partner activates the MgATPase activity of FLM7a in the presence of low concentrations of actin. We are currently mapping the areas on FLM7a that interact with the binding partner and vice versa. We find that a GFP-tagged full length myosin VIIa (GFP-FLM7a) has a diffuse localization when expressed in Drosophila S2 cells in culture. The same is true when an mCherry M7BP is expressed by itself in these cells. However, co-transfection of S2 cells with GFP-FLM7a and mCherry M7BP results in a marked activation on cellular cytoskeletal activity. The cells experience marked ruffling of the lamellipodia and grow numerous filopodia. FLM7a and M7BP are extensively co-localized in the regions of actin filament formation and are present along and at the tips of filopodia and can be observed moving together toward filopodial tips. Myosin VIIa and the M7BP are both expressed in hemocytes, a phagocytic cell type found in the hemolymph of larva which can phagocytize bacteria. Hemoctyes from flies that do not express myosin VIIa do not efficiently phagocytize bacteria. We have created a transgenic fly that expresses a GFP-tagged FLM7a and can observe the localization of the myosin when bacteria are being phagocytizedIn the larval eye disc, immunofluorescence staining of M7a, M7BP and Rab 11 are localized to the lens-secreting cone cells. EM sections of the adult eyes of the M7a mutants showed missing pigment granules surrounding the primary pigment cells and at the base of the rhabdomeres of the photoreceptor cells. Scanning EM of the eye of M7a mutant and M7BP mutant showed abnormal bristles. The M7BP mutant is a P-element insertion line and we are presently performing excision mutagenesis to isolate more M7BP mutants. . We are currently examining the mechanical ability of myosin VIIa truncations using optical trapping nanometry. Preliminary results reveal that this myosin has a long attachment lifetimes and a long power stroke.

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National Heart, Lung, and Blood Institute
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