Silvia R. da Costa

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.

Unique ultrastructure of exorbital lacrimal glands in male NOD and BALB/c mice.

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.

Cytoskeletal participation in stimulated secretion and compensatory apical plasma membrane retrieval in lacrimal gland acinar cells.

One of the main functions of the lacrimal gland is the regulated secretion of tear fluid, rich in proteins and electrolytes, in response to signals provided through the sympathetic and parasympathetic nervous system. Since proper tear-fluid secretion is essential for maintenance of ocular health, a long-term focus of our laboratory has been to understand the molecular mechanisms governing regulated secretion in lacrimal acini. In particular, we have focused on the role of microtubules (MTs), actin-based microfilaments (MFs), and motor proteins associated with either filament system in the stimulated lacrimal acinar secretory response. MTs and MFs constitute two of the three major cytoskeletal filament systems in mammalian cells, the third system being the intermediate filaments.

Changes in cytoskeletal organization in polyoma middle T antigen-transformed fibroblasts: involvement of protein phosphatase 2A and src tyrosine kinases.

The major transforming activity of polyomavirus, middle T antigen, targets several cellular regulatory effectors including protein phosphatase 2A and src tyrosine kinases. Although transformed cells exhibit profound morphological changes, little is known about how middle T antigen-induced changes in the cellular regulatory environment specifically affect the cytoskeleton. We have investigated these changes in 10T(1/2) mouse fibroblasts transformed with polyoma middle T antigen. Immunofluorescence microscopy revealed that expression of middle T antigen (Pym T cells) depleted the stable (acetylated) microtubule array and increased the sensitivity of dynamic (tyrosinated) microtubules to nocodazole-induced disassembly. These effects were associated with a modest but statistically significant (P</=0.05) increase in recovery of protein phosphatase 2A activity with microtubules. Middle T antigen expression also depleted the normal cellular complement of actin stress fibers and focal adhesions, in parallel with changes in the distribution of src tyrosine kinases. Herbimycin A promoted recovery of paxillin and phosphotyrosine into nascent focal adhesion sites, in addition to restoring normal src tyrosine kinase distribution. However, herbimycin A did not restore actin stress fibers or parental-type microtubules to Pym T cells. We suggest that regulation of the microtubule array by middle T antigen may occur through direct effects including redistribution of protein phosphatase 2A as well as indirect effects such as altered interactions with actin-based stress fibers.