C.M. Rose

Cytoplasmic dynein participates in apically targeted stimulated secretory traffic in primary rabbit lacrimal acinar epithelial cells

A major function of the acinar cells of the lacrimal gland is the production and stimulated release of tear proteins into ocular surface fluid. We investigate the participation of cytoplasmic dynein in carbachol-stimulated traffic to the apical plasma membrane in primary rabbit lacrimal acinar epithelial cells. Confocal fluorescence microscopy revealed a major carbachol-induced, microtubule-dependent recruitment of cytoplasmic dynein and the dynactin complex into the subapical region. Colocalization studies, sorbitol density gradient/phase partitioning analysis and microtubule-affinity purification of membranes showed that some dynein and dynactin complex were associated with VAMP2-enriched membranes. Adenovirus-mediated overexpression of p50/dynamitin inhibited the recruitment and colocalization of dynein, the dynactin complex and VAMP2 in the subapical region. Nocodazole treatment and p50/dynamitin overexpression also depleted subapical stores of rab3D in resting acini, suggesting that dynein activity was also involved in maintenance of rab3D-enriched secretory vesicles. These data implicate cytoplasmic dynein in stimulated traffic to the apical plasma membrane in these secretory epithelial cells.

Accumulation of Catalytically Active Proteases in Lacrimal Gland Acinar Cell Endosomes During Chronic Ex Vivo Muscarinic Receptor Stimulation

Chronic muscarinic stimulation induces functional quiescence (Scand J Immunol 2003;58:550–65) and alters the traffic of immature cathepsin B (Exp Eye Res 2004;79:665–75) in lacrimal acinar cells. To test whether active proteases aberrantly accumulate in the endosomes, cell samples were cultured 20 h with and without 10‐µm carbachol (CCh), incubated with [125I]‐bovine serum albumin and then lysed and analysed by subcellular fractionation. CCh decreased total cysteine protease and cathepsin S activities in the isolated lysosome, redistributing them to early endocytic and biosynthetic compartments. CCh decreased [125I] accumulation in all compartments of cells loaded in the absence of protease inhibitors; the cysteine protease inhibitor, leupeptin, prevented the endosomal decrease but not the lysosomal decrease. Sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and autoradiography demonstrated [125I]‐labelled proteolytic products in endomembrane compartments of both control and CCh‐stimulated cells, even in the presence of leupeptin, but analysis indicated that CCh increased the amount in endosomes. Two‐dimensional fractionation analyses suggest that the CCh‐induced redistributions result from blocks in traffic to the late endosome from both the early endosome and the trans‐Golgi network. Therefore, we conjecture that chronic muscarinic acetylcholine receptor stimulation leads to aberrant proteolytic processing of autoantigens in endosomes, from whence previously cryptic epitopes may be secreted to the underlying interstitial space.

Novel biphasic traffic of endocytosed EGF to recycling and degradative compartments in lacrimal gland acinar cells.

The purpose of this study was to delineate the traffic patterns of EGF and EGF receptors (EGFR) in primary cultured acinar epithelial cells from rabbit lacrimal glands. Uptake of [125I]‐EGF exhibited saturable and non‐saturable, temperature‐dependent components, suggesting both receptor‐mediated and fluid phase endocytosis. Accumulation of [125I] was time‐dependent over a 120‐min period, but the content of intact [125I]‐EGF decreased after reaching a maximum at 20 min. Analytical fractionation by sorbitol density gradient centrifugation and phase partitioning indicated that within 20 min at 37°C [125I] reached an early endosome, basal–lateral recycling endosome, pre‐lysosome, and lysosome. Small components of the label also appeared to reach the Golgi complex and trans‐Golgi network. Intact [125I]‐EGF initially accumulated in the recycling endosome; the content in the recycling endosome subsequently decreased, and by 120 min increased amounts of [125I]‐labeled degradation products appeared in the pre‐lysosomes and lysosomes. Confocal microscopy imaging of FITC‐EGF and LysoTrackerRed revealed FITC enriched in a dispersed system of non‐acidic compartments at 20 min and in acidic compartments at 120 min. Both confocal immunofluorescence microscopy and analytical fractionation indicated that the intracellular EGFR pool was much larger than the plasma membrane‐expressed pool at all times. Cells loaded with [125I]‐EGF released a mixture of intact EGF and [125I]‐labeled degradation products. The observations indicate that in lacrimal acinar cells, EGFR and EGF–EGFR complexes continually traffic between the plasma membranes and a system of endomembrane compartments; EGF‐stimulation generates time‐dependent signals that initially decrease, then increase, EGF–EGFR traffic to degradative compartments. J. Cell. Physiol. 199: 108–125, 2004© 2003 Wiley‐Liss, Inc.