Impairing actin filament or syndapin functions promotes accumulation of clathrin-coated vesicles at the apical plasma membrane of acinar epithelial cells.

In this article, we investigate the contributions of actin filaments and accessory proteins to apical clathrin-mediated endocytosis in primary rabbit lacrimal acini. Confocal fluorescence and electron microscopy revealed that cytochalasin D promoted apical accumulation of clathrin, alpha-adaptin, dynamin, and F-actin and increased the amounts of coated pits and vesicles at the apical plasma membrane. Sorbitol density gradient analysis of membrane compartments showed that cytochalasin D increased [14C]dextran association with apical membranes from stimulated acini, consistent with functional inhibition of apical endocytosis. Recombinant syndapin SH3 domains interacted with lacrimal acinar dynamin, neuronal Wiskott-Aldrich Syndrome protein (N-WASP), and synaptojanin; their introduction by electroporation elicited remarkable accumulation of clathrin, accessory proteins, and coated pits at the apical plasma membrane. These SH3 domains also significantly (p

Growth of purified lacrimal acinar cells in Matrigel raft cultures.

The objective of this study was to develop a tissue culture system which closely mimics the in situ lacrimal gland for improved study of lacrimal acinar cell physiology. Highly purified preparations of lacrimal acinar cells from adult female New Zealand White rabbits were isolated and grown in suspension culture in the form of Matrigel 'rafts', i.e., aggregates of acinar cells enclosed within a Matrigel coating. The rafts were seeded onto Matrigel-coated culture plates and their growth was followed for up to 28 days. Immunohistochemistry was used to demonstrate the cellular sites of prolactin (PRL), epidermal growth factor (EGF), basic fibroblast growth factor (FGF-2), secretory component (SC) and major histocompatibility complex class-II molecules (MHC-II) within the acinar cells. By 3 days the cultures contained numerous, well-formed acini enclosed within the Matrigel. The acinar epithelial cells demonstrated histotypic polarity, with large, pale-staining, secretory granules aggregated adjacent to the lumen, and exocytotic release of secretory material into the lumen. From 5-10 days the pale-staining secretory granules decreased in number, while the lumenal contents of the acini increased in staining density. Throughout the culturing period as the pale-staining, secretory granules decreased in number, smaller more densely stained, secretory granules increased in number. The number of cells and size of acinar clusters increased steadily throughout the culturing period, and acini frequently achieved dimensions in excess of 0.5 mm. Increases in the size of acinar clusters were often accompanied by an increase in the size of the lumen. Frequently the lumen and its contents bulged asymmetrically towards one edge of the acinus. Immunhistochemistry demonstrated PRL and EGF within the lumens and within the apical cytoplasm of the acinar cells. Acini were strongly immunopositive for SC throughout the 28 day culture period, whereas immunopositivity for MHC-II molecules was strong initially, but diminished dramatically by 21 days. Immunostaining for FGF-2 was most intense on days 1 and 3, with staining throughout the cytoplasm, but became progressively more localized to the periphery of the acini as the culture period lengthened. In cultures of 1-28 days duration, Western blots of cell lysates demonstrated a major band (approximately 40 kDa) for PRL in 3-28 day preparations; a major band (approximately 80 kDa) for SC in 3 day and 7 day preparations that decreased in intensity in 14-28 day preparations; and a major band (approximately 23 kDa) for MHC-II protein in 1-21 day preparations that decreased in intensity in 28 day preparations. Lysosomes increased in number with time in culture, becoming a dominant cytoplasmic feature in 21 and 28 day cultures. Carbachol stimulation of 4 day rafts resulted in increased release of beta-hexosaminidase and SC from the rafts. The authors conclude that Matrigel rafts containing purified lacrimal gland acinar cells offer a highly advantageous system for study of lacrimal acinar cell function and one that correlates well with the in situ gland.