Basal-Lateral/Endomembrane Traffic in Lacrimal Acini

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DESCRIPTION (provided by applicant): Sjogren's syndrome (SjS) and other immune-related diseases cause lacrimal dysfunction, impaired vision, and ocular surface inflammation in 2 to 4 million Americans. Primary lacrimal deficiency (PLD), which also may have an immune-related component, affects at least 6 million more. These concepts are unified by the hypothesis that: the milieu in the lacrimal glands reflects an immunohomeostasis involving regulatory lymphocytes, autoimmune effector lymphocytes, and secretory epithelial cells; lymphocyte and inflammatory cell mediators modulate epithelial cell functions; and the immunohomeostasis evolves in response to altered epithelial cell functions. However, little is known about the triggers that alter epithelial function to initiate the diseases, the reasons they occur more frequently in women than in men, and the molecular mechanisms that cause lacrimal dysfunction. The investigators have found that chronic stimulation of lacrimal acinar cells with the muscarinic receptor (MAChR) agonist, carbachol, causes epithelial secretory quiescence and also activates an aberrant endomembrane traffic program that blocks movement to lysosomes, potentially increasing exposure of constitutive autoantigens and initiating exposure of previously cryptic epitopes. The G proteins that classically couple to MAChR, Gq and G11, are shared by receptors for a wide variety of mediators, and their signaling is influenced by the sex hormones. Therefore, the investigators propose that: (a) physiological perturbations can initiate inappropriate Gq/G11 signaling that activates the aberrant traffic program, and (b) the local environments in SjS and PLD stimulate inappropriate Gq/G11 signaling that causes functional quiescence underlying lacrimal dysfunction. Specific Aim 1. What are the signals that activate the aberrant membrane traffic program? The central hypothesis is that MAChR remain activated and continue to activate Gq/G11. Specific Aim 2. Can chronic stimulation of other receptors also cause functional quiescence and activate the aberrant traffic program? The central hypothesis is that these changes can be elicited by agonists for receptors that utilize Gq/G11, including histamine, 5-hydroxytryptamine, PGE2, and estradiol. Specific Aim 3. What traffic effectors are responsible for the aberrant program? The central hypothesis is that chronic stimulation decreases dynein motor function but increases p150(Glued) association with kinesin II, thereby increasing kinesin-mediated traffic. Specific Aim 4. How extensively does the aberrant program alter traffic of lysosomal proteins? The investigators will use a GFP-cathepsin S fusion protein and confocal microscopy to test the hypothesis that lysosomal proteins accumulate in the endosomes.