Methyl-ß-Cyclodextrin Impairs Apical Clathrin-Mediated Endocytosis in Lacrimal Acini

Abstract

Abstract: : Purpose: We have previously demonstrated that carbachol (CCH) stimulation of lacrimal acini promotes endocytic retrieval of secretory vesicle membrane from the apical surface, a process which utilizes clathrin and associated proteins, actin filaments and syndapins. Previous work in cell lines has demonstrated that cholesterol is essential for formation of clathrin-coated endocytic vesicles. Here we investigate the contribution of cholesterol to this process, using methyl-ß-cyclodextrin (MBCD) to deplete cholesterol. Methods: Rabbit lacrimal acini cultured for 2-3 days were treated with or without MBCD (1 mM, 30-150 min) in the presence and absence of CCH stimulation (100 µM, 15 min). Clathrin and α-adaptin localization were monitored by confocal fluorescence microscopy using appropriate primary and fluorescent secondary antibodies or affinity label. Endocytic activity was measured by FITC-dextran uptake and FACS analysis, or by ¹²⁵I- BSA uptake and gamma-counting. Subcellular membrane compartments from resting and CCH-stimulated acini with and without MBCD were isolated by centrifugation over sorbitol density gradients and the distributions of proteins of interest analyzed by Western blotting. Results: Confocal fluorescence microscopy revealed that α-adaptin and clathrin immunoreactivity were detected in a punctate pattern in resting acini with and without MBCD, but that MBCD increased their accumulation at the apical membrane after CCH stimulation. Previous data suggests that CCH-stimulated uptake of FITC-dextran occurs primarily at the apical membrane. MBCD did not reduce unstimulated uptake of FITC-dextran or ¹²⁵I-BSA, but it prevented the significant accumulation of FITC-dextran caused by CCH stimulation, and reduced the uptake of ¹²⁵I-BSA in the presence of CCH. Analysis of clathrin distribution across subcellular membrane compartments isolated over density gradients suggest that CCH-stimulation results in redistribution of clathrin to trans-Golgi network membranes following the apical budding and internalization of apical clathrin-coated pits. MBCD prevented this CCH-induced redistribution of clathrin to the trans-Golgi network, substantiating its inhibitory effect on apical clathrin-mediated endocytosis. Conclusion: MBCD-induced depletion of cholesterol from lacrimal acinar membranes impairs clathrin-mediated retrieval from the apical membrane.

Keywords: lacrimal gland • cell membrane/membrane specializations • microscopy: confocal/tunneling