Altered traffic to the lysosome in an ex vivo lacrimal acinar cell model for chronic muscarinic receptor stimulation.

Evidence suggests that lacrimal and salivary epithelial cells constitutively expose potentially pathogenic autoantigens, but that active regulatory networks normally suppress pathological autoimmune responses . Events that potentially disrupt the regulatory networks include increased exposure of constitutive autoantigens and induced exposure of previously cryptic autoantigen epitopes. Chronic muscarinic receptor (MAChR) stimulation in an ex vivo rabbit lacrimal acinar cell model induces functional and biochemical alterations reminiscent of the functional quiescence associated with Sjogren's syndrome . Chronic MAChR stimulation also elicits changes in the compartmental distribution of beta-hexosaminidase, a product that normally is dually targeted into the lysosomal pathway and the regulated apical secretory pathway. Here, we use subcellular fractionation analyses to further explore the nature of the stimulation-induced traffic changes and to identify effectors that might mediate this change. Overnight stimulation of primary cultured rabbit lacrimal gland acinar cells with 10 microM carbachol (CCh) significantly decreased the abundance of mature cathepsin B in the pre-lysosome and lysosome; decreased the abundance of preprocathepsin B in fractions containing the TGN and late endosome; increased the abundance of procathepsin B in fractions containing the basal-lateral membrane; and increased the accumulation of endocytosed [(125)I]-EGF in the recycling endosome. Alterations in distribution or abundance of traffic effectors included: increased abundances of rab5A and rab6 in the TGN; decreased overall abundance of gamma-adaptin; remarkably increased relative abundance of membrane phase-associated actin; redistribution of cytoplasmic dynein from biosynthetic and proximal endocytic compartments to the lysosome; and redistribution of p150(Glued) from the lysosome to biosynthetic or proximal endocytic compartments. We conclude that chronic MAChR stimulation blocks traffic from the early endosome and the TGN to the lysosome, causing lysosomal proteins to reflux to the TGN, endosomes, and basal-lateral membrane. These traffic alterations may be mediated through action on one or more of the effectors noted.

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 [(125)I]-EGF exhibited saturable and non-saturable, temperature-dependent components, suggesting both receptor-mediated and fluid phase endocytosis. Accumulation of [(125)I] was time-dependent over a 120-min period, but the content of intact [(125)I]-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 degrees C [(125)I] 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 [(125)I]-EGF initially accumulated in the recycling endosome; the content in the recycling endosome subsequently decreased, and by 120 min increased amounts of [(125)I]-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 [(125)I]-EGF released a mixture of intact EGF and [(125)I]-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.

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.