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S. Xu, L. Chiang, A. K. Mircheff, C. T. Okamoto, S. F. Hamm-Alvarez; Characterization of Rab11a-Enriched Membrane Compartments In Lacrimal Gland Acinar Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5195.
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© ARVO (1962-2015); The Authors (2016-present)
Lacrimal gland acinar cells (LGACs) are secretory epithelial cells. In other epithelia, Rab11a is associated with endocytic membrane compartments and is thought to participate in endocytosis and transcytosis. The goal of this project was to determine the function of Rab11a in LGACs.
For biochemical studies, primary cultured LGACs were homogenized and resultant subcellular membranes were fractionated on discontinuous sucrose density gradients under ultracentrifugation. Fractions were probed for membrane compartment markers by Western blotting. For live and fixed cell imaging studies, LGACs were transduced with Adenovirus (Ad) constructs expressing GFP-Rab11a and/or mCherry-Myosin Vb prior to analysis by confocal microscopy. Tetramethylrodamine (TMR)-conjugated Dextran 70K and Rab3D were used as fluid-phase endocytosis and secretory vesicle markers, respectively.
Membrane compartments were partially resolved by the discontinuous sucrose density gradient: early endosomes, Rab3D-enriched secretory vesicles and Rab11a-enriched membranes were concentrated at the supernatant/24% sucrose interface; Rab7-enriched late endosomes were concentrated in the 24%/37% sucrose interface; and lysosomes were concentrated in the 37%/60% sucrose interface. By confocal microscopy, GFP-Rab11a-enriched vesicles were localized near the apical membrane and exhibited dynamic tubulovesicular movements that were enhanced by carbachol. Co-expression of mCherry-Myosin Vb tail completely abolished movement of GFP-Rab11a-enriched vesicles. GFP-Rab11a was completely co-localized with mCherry-Myosin Vb tail, while it exhibited only partial localization with Rab3D and YFP-Rab27b. However, GFP-Rab11a-enriched vesicles were not accessible to endocytosed TMR-Dextran 70K.
Rab11a-enriched membranes overlapped with both endosomes and secretory vesicles on fractions of discontinous sucrose gradients, while their subapical enrichment and significant colocalization with Myosin Vb tail but not Rab3D observed by confocal microscopy suggested association with apical recycling endosomes. However, the lack of access of TMR-Dextran to Rab11a-enriched compartments suggests that this apical compartment in LGAC is not involved in fluid-phase endocytosis and may be highly specialized for the trafficking of certain cargo.
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