Role of Rab3A in regulated secretion

• Main Authors: Kuo-Heng Cheng, 鄭國亨
• Other Authors: Lung-Sen Kao
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/46824590050565634847

碩士 === 國立陽明大學 === 生物化學研究所 === 93 === Neurons possess two types of secretory vesicles which are distinguished by the morphological appearance: large dense-core vesicles (LDCVs), and small synaptic vesicles (SSVs). In my study, EGFP-tagged neuropeptide Y (NPY-EGFP), a soluble protein in the vesicles, and synapto-pHluorin (spH), a fusion protein of VAMP2 and pHluorin, were used as markers of LCDVs and SSVs, respectively. Results from immunocytochemistry and fluorescence lifetime imaging microscopy (FLIM) experiments were shown that NPY and spH seemed distinct subcellular localizations in rat pheochromocytoma PC12 cells. In previous studies, Rab3A, one of the small GTP-binding proteins, has been implicated in Ca2+-dependent exocytosis. Results from recent studies support that Rab3A is involved in docking, priming and fusion steps of secretory pathway, and exerts its action by interacting with several effectors, such as Rabphilin3A, calmodulin, and RIM. However, the precise steps in regulated secretion that Rab3A regulates remain incompletely understood. In my study, various ECFP-tagged Rab3A mutants (ECFP-Rab3A) and NPY-EGFP were co-transfected in PC12 cells. Various Rab3A mutants were used to study the role of RAb3A in processes of exocytosis. NPY-EGFP was used as a marker for secretory vesicles. Total internal reflection fluorescence microscopy (TIRFM) was used to monitor the fluorescence changes of NPY-EGFP during exocytosis. The fluorescence of NPY-EGFP in PC12 cells exhibited a punctuate pattern, which was partial co-localized with chromogranin(A+B) that was thought to represent the dense core vesicles. Upon ATP stimulation, the NPY-EGFP fluorescence increased first and then emerged in a cloud and rapidly diffused away. When a constitutive active GTP-bound mutant of Rab3A, Rab3A Q81L, was overexpressed, it caused slower rate and narrow range of movement, and longer retention of NPY-EGFP-labeled vesicles around plasma membranes upon stimulation. Overexpression of a Rab3A F59S mutant, which was defective in the interaction with rabphilin3A, or of a Rab3A R66L-R70T mutant, which was defective in binding with calmodulin, caused a slow release of NPY-EGFP-labeled vesicles upon stimulation. In general, the NPY-EGFP fluorescence increased transiently and then dimmed during exocytosis. However, over half of vesicles were no transient increase in cells expressing a GDP form mutant of Rab3A, Rab3A T36N. It is speculated that the pattern of the transient increase of NPY-EGFP fluorescence during exocytosis may be associated with the priming step before fusion. Because of pH sensitivity of EGFP protein, it was also demonstrated that the transient increase of NPY-EGFP fluorescence did not result from changes in the intravesiclular pH during exocytosis. In summary, these data provide a direct evidence that Rab3A plays an important role in the late step in exocytosis.