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Plasma Membrane Internalization and Recycling in Rabbit Lacrimal Acinar Cells

By Robert W. Lambert, F Carol A. Maves and Austin K. Mircheff


Purpose. The purpose of this study was to examine internalization and recycling of plasma membrane constituents in lacrimal gland acinar cells. Methods. Acinar cells were isolated from rabbit lacrimal glands. Surface-expressed reactive groups were biotinylated at 4°C with sulfo-N-hydroxysuccinimidyl-biotin. Incorporated biotin was then labeled with avidin-horseradish peroxidase complex for light microscopy, with avidinlucifer yellow conjugate for fluorescence microscopy, and quantitative fluorometry, and with avidin-ferritin conjugate for electron microscopy. Results. At 4°C labels remained at the surfaces of intact cells. Surface avidin-lucifer yellow decreased markedly, giving way to punctate cytoplasmic labeling, on warming to 37°C. Electron microscopy of cells warmed after labeling with avidin-ferritin revealed ferritin in smooth vesicles underlying the plasma membranes, in vesicles adjacent to Golgi membranes, and in multivesicular bodies. Incubation at 37°C before chilling and labeling with avidin-lucifer yellow decreased the cells ' capacity to bind avidin-lucifer yellow by 95%, with t05 < 0.5 min. If cells were then incubated with avidin-lucifer yellow at 37°C, they took up the marker with a time course that indicated that 60 % of the initial biotin either recycled back to the plasm

Year: 2013
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