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G.C. Munguba, M. Jackson, M. Hunt, G. Zimowska, S.P. Sugrue; Pinin Interacts With Keratin at Cell Adhesion Sites and SR-Proteins in Nuclear Speckles in Corneal Epithelial Cells (HCE-T) . Invest. Ophthalmol. Vis. Sci. 2003;44(13):924.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the means and regulation of cell-cell adhesion of corneal epithelium we have focused our investigations on a epithelial cell adhesion- related and nuclear protein named pinin (Pnn). We suggest that the regulation of Pnn is a key event in cell-cell adhesion during epithelial migration. This study is proposed to verify the co-localization of protein partner(s) of pinin in corneal epithelial cells. Previously, we employed the yeast two-hybrid approach to identify potential interactions between Pnn and other proteins. We showed that Pnn is capable of binding to keratins and multiple nuclear proteins such as SR-rich proteins including a novel SRrp130, SRp75, and SRm300. Here we wish to confirm these interactions in the context of corneal epithelial cells. Methods: Cultured human corneal epithelial cells (HCE-T, RCB1384, K. Sasaki) were immunostained with specific antibodies to Pnn, keratin, plakins, SRm300, Srp75, and SR proteins. Cells were transfected with full length, truncated, and tagged portions of Pnn, SRm300, SRp75, SRrp130. Results: Immunostaining of HCE-T cells revealed that Pnn was localized to points of keratin filament convergence to lateral membrane at cell-cell adhesion sites co-localizing with desmoplakin and within the nucleus distributed both in speckles and diffusely throughout intrachromatin space. Transfections of HCE-T cells with full length, truncated, and tagged portions of Pnn, SRm300, SRp75, SRrp130 demonstrated co-distribution of Pnn with SRm300, SRp75 and SRp130. Interestingly, co-transfection with SRp75 caused Pnn to distribute more diffusely, while co-transfections with SRp130 caused Pnn to form large spheroid-like speckles. Conclusions: The data indicate that in corneal epithelial cells, like the HeLa cell, Pnn is found within the nucleus as well as with keratin filaments at cell-cell adhesion sites. These results also suggest that Pnn may participate in a multi-protein nuclear complex, that includes SRp75, SRm300 and SRp130 and is located at the nuclear speckle. (Supported by NIH grant EY07883 )
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