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R. Maddala, J. Qiu, P. Vasantha Rao; Mouse Lens Fiber Cell Elongation and Differentiation Is Associated With Increased Myosin II Phosphorylation . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2938.
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© ARVO (1962-2015); The Authors (2016-present)
Myosin II, a molecular motor protein, plays a critical role in the processes of cell migration, cell morphology, morphogenesis and cytokinesis. To understand the potential involvement of Myosin II in lens fiber cell elongation and differentiation, we determined the distribution pattern of myosin II isoforms and its phosphorylation status in the developing mouse lens.
The distribution pattern of non–muscle myosin IIA, IIB and phosphorylated regulatory myosin light chain (phospho–MLC) in the developing mouse lens was determined by immunofluorescence confocal imaging. In situ Rho GTPase activation in the developing lens was analyzed using a Rhotekin–Rho–GTP binding assay to evaluate whether myosin II phosphorylation and Rho GTPase activation were associated. Lenses obtained from transgenic mice expressing either the C3–exoenzyme or RhoGDI in a lens specific manner were evaluated to assess the role of Rho GTPase function in regulating phosphorylation of lens myosin II. The effects of myosin II inhibition on actin cytoskeletal organization, cell adhesions and adherens junctions were tested in mouse lens primary epithelial cells by immunofluorescence.
While myosin IIA is distributed uniformly throughout the developing lens including the epithelium and differentiated fibers, myosin IIB was localized strongly to the epithelium. On the other hand, phospho–MLC localizes intensely and somewhat specifically to the elongating and differentiating lens fibers. Further, phospho–MLC exhibited a clear co–localization with actin cytoskeleton labeled with phalloidin, in differentiating fibers but not in the epithelium. Although, the distribution of Rho GTPase and its in situ activation analysis revealed its distribution and activation throughout the developing lens including lens epithelium and lens fibers, lens specific Rho GTPase inactivation by the tissue specific expression of C3–exoenzyme and Rho GDI was found to suppress MLC phosphorylation and disrupt actomyosin organization in lens fibers. Myosin IIA and IIB exhibited differential distribution patterns in cultured mouse lens primary epithelial cells and treatment with blebbistatin (myosin II inhibitor) was noted to affect the cell shape, cell adhesions and cell–cell junctions in these cells.
Taken together, this data reveal a distinct spatial distribution pattern of non–muscle myosin II isoforms in lens and an association between increased myosin II phosphorylation regulated by Rho GTPases and lens fiber cell elongation and differentiation.
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