Myosin 7a 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 previously shown that Drosophila myosin 7a that 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 7a(GFP-FLM7a) has a diffuse localization when expressed in Drosophila S2 cells in culture. The same is true when a 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. This suggests that M7BP may dimerize myosin 7a and allow it to be processive. Consistent with this, while myosin 7a alone does not move processively on actin filaments in a single molecule motility assay, it does move processively when M7BP is also added to the assay. The velocity is very slow, but the run length is around 0.5 um. Thus the residence time that the myosin-7a/M7BP complex spends on actin is very long. The most predominate translocating particle has two myosin 7a motor domains, suggesting that M7BP dimerizes the molecules. Myosin 7a and the M7BP are both expressed in larval hemocytes, which are macrophage-like cells found in the hemolymph of larva and have the ability to phagocytose bacteria. Hemocytes from flies that do not express myosin VIIa do not efficiently phagocytose 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 phagocytosed. In 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 also showed abnormal bristles. When we knockdown either M7a or M7BP in the pigment cells in the eye using RNAi lines, we observed roughness of the eye, suggesting that both M7a and M7BP interacts within the same pathway. We have also made full length Drosophila myosin 5 and are studying the basis for the regulation of this molecule.
| Haithcock, Jessica; Billington, Neil; Choi, Kevin et al. (2011) The kinetic mechanism of mouse myosin VIIA. J Biol Chem 286:8819-28 |
| Billington, Neil; Sellers, James R (2011) Dynein struts its stuff. Nat Struct Mol Biol 18:635-6 |
| Siththanandan, Verl B; Sellers, James R (2011) Regulation of myosin 5a and myosin 7a. Biochem Soc Trans 39:1136-41 |
| Yang, Yi; Baboolal, Thomas G; Siththanandan, Verl et al. (2009) A FERM domain autoregulates Drosophila myosin 7a activity. Proc Natl Acad Sci U S A 106:4189-94 |
