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C.–Y. Chen, K. Wu, M. MacVeigh, M. Pidgeon, J. Schechter, S.F. Hamm–Alvarez; Rab3d Knockout Mouse Lacrimal Glands Exhibit Secretory Vesicles with Increased Vesicle Diameter and Aberrant Morphology . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1951.
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© ARVO (1962-2015); The Authors (2016-present)
Rab3D is a member of the secretory Rab3 subfamily of Rab proteins which each facilitate different steps in intracellular membrane trafficking. Rab3D is enriched on mature secretory vesicles (SVs) in acinar epithelial cells from pancreas, parotid gland and lacrimal gland but its function in formation and/or exocytosis of SVs is unknown. Previous studies in Rab3D knockout (KO) mice showed a significant increase in SV diameter in pancreas and parotid gland compared to the same organs in control (C57BL/6) mice (Riedel et al, Mol Cell Biol 22:6487, 2002). The purpose of this study was to determine whether loss of rab3D similarly affected SVs in lacrimal gland, and to discern aspects of its function in exocytosis.
Sections from LG from 4 week Rab3D KO and C57BL/6 mice were processed for electron microscopy (EM) evaluation of SV diameter and morphology, and for confocal fluorescence microscopy evaluation of rab3D and actin filaments. Results are from 1–2 mice per condition.
The average SV diameter in LG acinar cells from the Rab3D KO mouse was significantly larger (p≤0.05) than that for SVs in sex–matched C57BL/6 mouse LG acinar cells. Preliminary findings suggested that the average SV diameter in LG acinar cells from male and female mice in each category also differed, with males exhibiting substantially smaller SVs. Specifically, female Rab3D KO mice exhibited LG SVs with a diameter of 1.44 ± 0.02 µm (n=396 SVs analyzed) while female C57BL/6 mice exhibited LG SVs with a diameter of 0.93 ± 0.02 µm (n=645 SVs). Male Rab3D KO mice exhibited LG SVs with a diameter of 0.97 ± 0.02 µm (n=602 SVs), while male C57BL/6 mice exhibited LG SVs with a diameter of 0.70 ± 0.01 µm (n=622 SVs). SVs in Rab3D KO mouse LGs exhibited a further morphological change in which SVs could be observed pinching off pieces of adjacent SVs by envelopment of a piece of the donor vesicle by the acceptor vesicle. Confocal fluorescence microscopy verified the loss of rab3D in the Rab3D KO mouse LGs and showed no evidence of altered actin cytoskeleton, a participant in apical exocytosis.
These findings suggest that Rab3D regulates intracellular homotypic fusion of LG SVs, and further suggest possible sex–based differences in SV size in this mouse strain.
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