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SR Da Costa, FA Yarber, M Kessels, B Qualmann, SF Hamm-Alvarez; A Role for Syndapin I and II and Mammalian Abp 1 in Clathrin-Mediated Apical Endocytosis in Rabbit Lacrimal Acinar Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3142.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Our previous work has implicated actin filaments in apical endocytosis in lacrimal acini. Studies in other systems have identified syndapins I and II and actin binding protein 1 (Abp1) as candidates for modulating the interactions between actin filaments and the endocytic machinery. Here we investigate their role in apical endocytosis in lacrimal acini. Methods: SH3 domains of syndapins I and II and of Abp1 were fused to glutathione-S-transferase (GST) and fusion proteins were expressed in E. coli and introduced by electroporation into three day primary cultures of rabbit lacrimal acinar cells. The location of clathrin, α-adaptin and dynamin was monitored by confocal fluorescence microscopy using appropriate primary and secondary antibodies. Pulldown assays with GST agarose followed by Western blotting were utilized to identify acinar proteins interacting with recombinant SH3 domains. Results: Introduction of GST-SH3 domains of syndapins I and II and of Abp1, but not GST or the mutated SH3 domain of syndapin I, into lacrimal acini resulted in a major enrichment of clathrin, α-adaptin and dynamin at the apical surface. The SH3 domains of syndapins I and II and of Abp1 also enhanced the formation of novel actin-coated invaginations at the apical surface which were decorated with clathrin, α-adaptin and dynamin. GST pulldown assays revealed that GST-SH3 domains of syndapins I and II and of Abp1, but not GST or the mutated SH3 domain of syndapin I, specifically bound dynamin. Measurements of basal and carbachol-stimulated (100 µM) protein and ß-hexosaminidase secretion revealed that the apical accumulation of clathrin, α-adaptin and dynamin caused by the SH3 domains of syndapins I and II and Abp1 did not decrease the exocytic response. Conclusion: Syndapins I and II and Abp1 participate in actin-mediated apical endocytosis in lacrimal acini and may also aid in remodeling of the apical actin network following exocytosis.
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