ABSTRACT
The acinar epithelial cells of the lacrimal gland are responsible for the production, packaging and regulated exocytosis of tear proteins into ocular surface fluid. This review summarizes new findings on the mechanisms of exocytosis in these cells. Participating proteins are discussed within the context of different categories of trafficking effectors including targeting and specificity factors (rabs, SNAREs) and transport factors (microtubules, actin filaments and motor proteins). Recent information describing fundamental changes in basic exocytotic mechanisms in the NOD mouse, an animal model of Sjögren's syndrome, is presented.
Subject(s)
Exocytosis/physiology , Lacrimal Apparatus/metabolism , Secretory Vesicles/metabolism , Actin Cytoskeleton/metabolism , Actins/metabolism , Animals , Cytoskeleton/metabolism , Disease Models, Animal , Eye Proteins/metabolism , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Lacrimal Apparatus/ultrastructure , Mice , Mice, Inbred NOD , Microtubules/metabolism , Models, Biological , Myosins/metabolism , Rabbits , SNARE Proteins/metabolism , Sjogren's Syndrome/metabolism , Vesicular Transport Proteins/metabolism , rab GTP-Binding Proteins/metabolismABSTRACT
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.
Subject(s)
Autoantigens/metabolism , Cell Membrane/metabolism , Epithelial Cells/metabolism , Lacrimal Apparatus/metabolism , Lysosomes/metabolism , Actins/metabolism , Animals , Biological Transport , Biomarkers/analysis , Carbachol/pharmacology , Cathepsin B/metabolism , Cell Fractionation , Cholinergic Agonists/pharmacology , Dyneins/metabolism , Epidermal Growth Factor/metabolism , Female , Models, Animal , Rabbits , Receptors, Muscarinic/drug effects , Stimulation, Chemical , alpha-Glucosidases/metabolismABSTRACT
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