Limin Qian

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.

Accumulation of Catalytically Active Proteases in Lacrimal Gland Acinar Cell Endosomes During Chronic Ex Vivo Muscarinic Receptor Stimulation

Chronic muscarinic stimulation induces functional quiescence (Scand J Immunol 2003;58:550–65) and alters the traffic of immature cathepsin B (Exp Eye Res 2004;79:665–75) in lacrimal acinar cells. To test whether active proteases aberrantly accumulate in the endosomes, cell samples were cultured 20 h with and without 10‐µm carbachol (CCh), incubated with [125I]‐bovine serum albumin and then lysed and analysed by subcellular fractionation. CCh decreased total cysteine protease and cathepsin S activities in the isolated lysosome, redistributing them to early endocytic and biosynthetic compartments. CCh decreased [125I] accumulation in all compartments of cells loaded in the absence of protease inhibitors; the cysteine protease inhibitor, leupeptin, prevented the endosomal decrease but not the lysosomal decrease. Sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and autoradiography demonstrated [125I]‐labelled proteolytic products in endomembrane compartments of both control and CCh‐stimulated cells, even in the presence of leupeptin, but analysis indicated that CCh increased the amount in endosomes. Two‐dimensional fractionation analyses suggest that the CCh‐induced redistributions result from blocks in traffic to the late endosome from both the early endosome and the trans‐Golgi network. Therefore, we conjecture that chronic muscarinic acetylcholine receptor stimulation leads to aberrant proteolytic processing of autoantigens in endosomes, from whence previously cryptic epitopes may be secreted to the underlying interstitial space.

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 [125I]‐EGF exhibited saturable and non‐saturable, temperature‐dependent components, suggesting both receptor‐mediated and fluid phase endocytosis. Accumulation of [125I] was time‐dependent over a 120‐min period, but the content of intact [125I]‐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°C [125I] 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 [125I]‐EGF initially accumulated in the recycling endosome; the content in the recycling endosome subsequently decreased, and by 120 min increased amounts of [125I]‐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 [125I]‐EGF released a mixture of intact EGF and [125I]‐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. J. Cell. Physiol. 199: 108–125, 2004© 2003 Wiley‐Liss, Inc.

Diverse Perturbations May Alter the Lacrimal Acinar Cell Autoantigenic Spectra

Lacrimal gland acinar cell autoantigens in Sjögrens syndrome include both intracellular proteins and plasma membrane proteins, to which the immune system normally must be tolerant. Attention has largely focused on the roles apoptotic cell death may play in exposing sequestered autoantigens and novel surface epitopes. We hypothesize that perturbations of ongoing membrane traffic in intact, functioning cells may also increase autoantigen exposure. We review the vesicular traffic between acinar cell basal-lateral plasma membranes (blm) and endomembrane compartments, then describe experiments in which isolated acinar cells were stimulated with epidermal growth factor (EGF), lysed, and analyzed by sorbitol gradient centrifugation. Whereas the cholinergic agonist, carbachol, impairs traffic from the trans-Golgi network to prelysosomes, causing Golgi, secretory, and lysosomal proteins to reflux into domains of the trans-Golgi network that communicate with the blm and to accumulate in the blm, EGF specifically causes a 2.6-fold (P < 0.05) increase in the beta-hexosaminidase content of the blm fraction, apparently by impairing traffic from early endosomes to prelysosome. We, therefore, suggest that a variety of physiologic stimuli may alter the spectra of autoantigens acinar cells secrete to the interstitium, express in their blm, and present via MHC Class II molecules after proteolytic processing.

M3 receptor autoimmunity: losing tolerance to a familiar protein.

The sera of Sjogren’s syndrome patients frequently contain autoantibodies to proteins normally localized in intracellular compartments. These include the Ro/SSA and La/SSB ribonucleoproteins, which are expressed in the nuclei; fodrin, which mediates interactions between the cytoskeleton and plasma membrane, and as-yet-uncharacterized proteins that appear to be associated with the Golgi complex. Theories to explain the loss of tolerance to the classical Sjogren’s autoantigens generally begin with the premise that under normal circumstances they are exposed to the immune system at levels so low that tolerance can be maintained through peripheral mechanisms.

Epidermal Growth Factor Traffic in Lacrimal Acinar Cells

Lacrimal gland acinar cells furnish a constitutive network of membrane vesicles between their basal-lateral plasma membranes and endomembrane compartments. This basal-lateral/endomembrane traffic may mediate normal acinar cell functions. However, it may also provide pathways for aberrant plasma membrane expression and secretion to the interstitium of autoantigen proteins. Additionally, it may be the source of aberrant MHC Class II molecules that mediate the presentation of processed autoantigen peptides. These phenomena could overwhelm peripheral tolerance mechanisms and promote the autoimmune responses of Sjogren’s syndrome.

IL-2 Immunoreactive Proteins in Lacrimal Acinar Cells

Sjogren’s syndrome is an autoimmune disease primarily involving the lacrimal and salivary glands. Recent studies on the initiation of autoimmune dacryoadenitis suggest that when acinar cells from rabbit lacrimal glands are induced to express MHC Class II molecules, the ability to present processed autoantigen peptides to CD4 T cells is also induced (Guo et al., 2000). However, it is likely that the outcome of MHC Class II molecule-mediated autoantigen presentation depends on the accessory signals present in the interstitial milieu. One potential accessory factor is interleukin 2 (IL-2), which is classically produced by Thl CD4 cells.

Glycolipid-rich Membrane Microdomains in Lacrimal Acinar Cell Endomembrane Compartments

Extensive molecular trafficking exists between the basal-lateral plasma membrane (blm) and endomembrane compartments of lacrimal gland acinar cells. Proteins normally must be segregated consistently between the lacrimal acinar cell’s apical secretory pathway, lysosomal pathway, and basal-lateral membrane recycling pathway. A recent analytical subcellular fractionation study (Yang et al., 1999b) showed that significant changes in the distributions of a variety of endomembrane proteins occur when lacrimal acinar cells are stimulated with 10 µM carbachol for 20 min. In that study, the amount of β-hexosaminidase, typically a lysosomal enzyme, but also a secretory enzyme in lacrimal acinar cells, increased in isolated low-density trans-Golgi network (ld-tgns) believed to represent domains of the trans-Golgi network that mediate traffic to the basal-lateral membrane. This increase was accompanied by net redistributions of protein into the ld-tgns and blm of proteins from other endomembrane compartments. These included galactosyltransferase from the Golgi complex, Rab6 from domains of the high-density trans-Golgi network (hd-tgns) that are believed to mediate traffic both to secretory vesicles and pre-lysosomes (preLys), and immature forms of cathepsin B from the hd-tgns and preLys. We have proposed that such alterations of protein segregation and targeting in the endomembrane traffic might overwhelm peripheral tolerance mechanisms and provoke local autoimmune responses (Mircheff et al., 1996, Mircheff et al., this volume).