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

The lacrimal glands of male NOD mice exhibit many of the features of the human lacrimal gland in patients afflicted with the autoimmune disease, Sjögren's syndrome, including loss of secretory functions and lymphocytic infiltration into the lacrimal gland. To elucidate the early changes in the secretory pathway associated with development of Sjögren's syndrome, we investigated the organization of the exocytotic pathway in lacrimal glands of age-matched male BALB/c and NOD mice. Cryosections from lacrimal glands from 1 and 4 month male BALB/c and NOD mice were processed for confocal fluorescence and electron microscopic evaluation of different participants in exocytosis. No changes in apical actin filaments were noted in glands from NOD mice, but these glands exhibited thickening of basolateral actin relative to that seen in the BALB/c mice. Rab3D immunofluorescence associated with mature secretory vesicles was distributed abundantly in a continuous vesicular network concentrated beneath the apical plasma membrane in glands from 1 and 4 month BALB/c mice. In glands from 1 month NOD mice, rab3D immunofluorescence exhibited marked discontinuity and irregularity in the vesicular labeling pattern. While this change was also detected in glands from 4 month NOD mice, many of these glands exhibited an additional extension of rab3D labeling through the cell to the basolateral membrane. Electron microscopic analysis confirmed the formation of irregularly-shaped, unusually large secretory vesicles in lacrimal glands from NOD mice. Quantitation of multiple secretory vesicles from electron micrographs revealed a significant (p ≤5) increase in the percentage of secretory vesicles incorporated into multivesicular aggregates in lacrimal glands from 1 and 4 month NOD mice compared to BALB/c mice. The M3 muscarinic receptor, a key signaling effector of exocytosis, was redistributed away from its normally basolateral locale in glands from BALB/c mice, with concomitant enrichment in intracellular aggregates in glands from NOD mice. These findings show that lacrimal glands in NOD mice as young as 1 month contain aberrant secretory vesicles with altered effector composition that undergo premature cytoplasmic fusion, and that changes in the distribution of the M3 muscarinic receptor occur within the same time frame.

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Lacrimal glands of male NOD and BALB/c mice have very small, pleomorphic acinar lumens. Acini contain isolated zones of highly complex cell surface interdigitations at the basal surface, sometimes occurring between acinar and myoepithelial cells. In NOD mice, cytological abnormalities, including mitochondrial deterioration, pleomorphic and heterogeneous cytoplasmic vacuoles, and lipid accumulation are evident within acinar cells at 1 month. Accumulation of lipid is further increased as the animal ages, accompanied by lymphocytic infiltration and destruction of acini. These results demonstrate alterations from normal cytology as early as 1 month in NOD mice, well before detection of clinical signs of Sjögren syndrome.

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