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 lenght molecule was expressed in Sf9 cells. We have examined the regulation of enzymatic activity of full length myosin VIIa and various C-terminally truncated fragments. Full length myosin VIIa (FLM7a) has a Vmax of about 1 per sec, but has a low apparent affinity for actin, requiring 30-50 uM acin for half-maximal activation. Various C-terminally truncated fragments have a similar Vmax, but much less actin is required for half maximal activation (0.5-1 uM). This means that at 5 uM actin the activity of the fragments is near maximal whereas that of the FLM7a is still barely activated. Removal of even the last 99 amino acids is sufficient to cause this remarkable change in activity. We explored the structural basis for this regulation by using single particle analysis in the electron microscope. We find that in the presence of ATP FLM7a is tightly folded into a compact structure such that the myosin motor domain cannot be discerned whereas in the absence of ATP the molecule is more extended and shows a clearly distinguishable motor domain. Removable of small bits of the tail also is accompanied by the extended conformation leading us to suggest that the compact structure represents an inhibited state of the molecule. Point mutations of a pair of conserved charged groups in the C-terminal tail also result in loss of regulation and unfolding of the molecule. We have expressed the second FERM domain of myosin VIIa and find that it can bind to actin in an ATP dependent manner. A 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 binding partner is expressed by itself in these cells. However, co-transfection of S2 cells with GFP-FLM7a and mCherry binding partner results in a marked activation on cellular cytoskeletal activity. The cells experience marked ruffling of the lamellipodia and grow numerous filopodia. FLM7a and the binding partner are extensively co-localized in the regions of actin filament formation and are present along and at the tips of filopodia. We are currently examining the mechanical ability of myosin VIIa using optical trapping nanometry. Preliminary results reveal that this myosin has a long attachment lifetime.
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