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L. Chiang, K. Wu, S.F. Hamm–Alvarez; Rab27 Isoforms Are Major Players in Lacrimal Gland Intracellular Trafficking . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3533.
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© ARVO (1962-2015); The Authors (2016-present)
Rab27 isoforms (a and b) participate in secretory membrane trafficking in diverse systems and have recently been implicated in the etiology of human diseases such as Hermansky–Pudlak syndrome and choroideremia. The purpose of this study was to determine whether these rab isoforms are expressed in lacrimal gland and also whether they participate in secretory membrane trafficking.
Primary rabbit lacrimal gland acinar cells (LGAC) with and without carbachol (CCH) stimulation were processed for confocal fluorescence microscopy utilizing appropriate primary and secondary antibodies to Rab27a, Rab27b and other membrane markers. Labeling patterns were confirmed in frozen sections from mouse LGAC. Rab27 isoforms were also confirmed in LGAC biochemically by Western blotting (WB) and immunoprecipitation and by RT–PCR.
Confocal fluorescence microscopy analysis as well as WB confirmed the presence of Rab27a and b isoforms in both rabbit and mouse LGACs, while RT–PCR confirmed expression in mouse LG. Analysis of the labeling pattern revealed by immunofluorescence showed punctate fluorescence at and beneath the lumenal region and in the perinuclear region. Quantitative co–localization analysis of confocal images (n=3–4) revealed a high degree of overlap between fluorescent pixels associated with labeling of Rab27 isoforms and several apically–targeted cargo proteins or apical compartment markers. For instance: 81±4% of total subapical Rab27b was co–localized with the polymeric IgA receptor (pIgR) while 76±4% of total subapical pIgR was co–localized with Rab27b; 75±1.5% of total subapical Rab27a was co–localized with the secretory vesicle marker, Rab3D while 72±2% of total subapical rab3D was co–localized with Rab27a; and 70±6% of total subapical Rab 27a was co–localized with the apical endosomal marker Rab11 and 65±11% of total subapical Rab11 was co–localized with Rab11. Stimulation with CCH slightly but consistently increased the co–localization of Rab27b with pIgR and Rab27a with Rab11, while slightly decreasing the co–localization of Rab27a with Rab3D. Stimulation with CCH also altered the intracellular distribution of Rab27a and Rab27b.
Both isoforms of Rab27 are expressed in mouse and rabbit LGAC. The relatively high co–localization coefficients with markers of the secretory and transcytotic pathways and the responsiveness to CCH suggest a role for these rabs in regulated trafficking to the apical plasma membrane.
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