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S. Xu, R. Marchelletta, L. Chiang, C. Okamoto, S. Hamm-Alvarez; Rab11a Distribution and Role in Trafficking of the Polymeric Immunoglobulin A Receptor in Primary Rabbit Lacrimal Gland Acinar Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4240.
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The polymeric immunoglobulin receptor (pIgR) mediates the basal-to-apical transcytosis of dimeric IgA across epithelial cells. pIgR expressed in lacrimal gland is responsible for the secretion of dimeric IgA into tears, an important element of ocular surface immune defense. Rab11a has been shown to participate in the transcytosis of pIgR in Madin-Darby canine kidney cells. The goal of this project was to determine the function of Rab11a in lacrimal gland, specifically in transcytosis of pIgR.
Primary cultured rabbit lacrimal gland acinar cells (LGACs) were co-transduced with Adenovirus (Ad) constructs encoding wild type (WT), constitutively active (CA) or dominant negative (DN) GFP-Rab11a in parallel with Ad encoding the Tet-on transactivator under the control of doxycycline. Co-transduction efficiency was approximately 90%. The distribution of the fusion protein was analyzed with direct and indirect fluorescence using confocal fluorescence microscopy. Protein expression was confirmed by Western blotting. In some cases, LGAC were treated with nocodazole (33 µM, 60 min) prior to analysis.
WT GFP-Rab11a was detected on intracellular vesicles that were mainly localized to the subapical region beneath the lumena, while DN GFP-Rab11a was extensively dispersed in the cytosol. The CA GFP-Rab11a was localized less to the subapical region than the WT construct and seemed to be partially dispersed in the cytosol. WT GFP-Rab11a showed high colocalization with subapical pIgR labeled by indirect immunofluorescence, while the DN GFP-Rab11a variant did not. Preliminary analysis using the weighted colocalization coefficient to quantify the extent of total WT GFP-Rab11a fluorescence co-localized with pIgR in individual cells revealed >70% of pixels were co-localized while the percentage of DN co-localized with pIgR dropped to <20%. The LGACs expressing WT GFP-Rab11a showed more pIgR localized to the subapical region than those expressing the DN variant. Moreover, disruption of the microtubule network by nocodazole dispersed most WT GFP-Rab11a subapical vesicles throughout the cytoplasm.
These data suggest that Rab11a participates in the apical targeting of pIgR, and that the GTP-binding state of Rab11a is critical for this activity. Furthermore, the subapical maintenance of Rab11a is dependent upon the microtubule array.
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