Researching of SNAP25-Rabphilin FRET Pair Within PC12 Cells by FLIM╱FRET

• Main Authors: Jiun-De Lee, 李俊德
• Other Authors: Yin Chang
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/66797649204078167675

碩士 === 國立陽明大學 === 生醫光電工程研究所 === 95 === Exocytosis, one kind of processes in membrane traffic, is proposed to contain four sequential steps: docking, priming, fusion, and recycling. While exocytosis is occurring, each step was regulated by the interaction between proteins and between protein and lipid. In terms of the technology of observing interaction between proteins, using Fluorescence Resonance Energy Transfer (FRET) into fluorescence microscopy is able to obtain approximate 200nm spatial resolution beyond the optics limit. Since the lifetime of fluorescent molecule will change during FRET, we used Fluorescence Lifetime Imaging Microscopy (FLIM) to observe both donor only (pEGFP-SNAP25 transfection) and coexistent of donor-acceptor (co-transfection of pEGFP-SNAP25 and mRFP-Rabphilin) ,and evaluated the difference in between to assure the existence of FRET phenomenon. Synaptosomal associated protein of 25 kDa (SNAP25), located in membrane protein, belongs to one type of Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE). According to the localization, SNAP25 is also termed to be the target-membrane associated protein (t-SNAREs). Rabphilin, the GTP-Rab3A specific binding protein, is also related to neurotransmitter and hormone secretion. It is first time that the interaction between SNAP25 and Rabphilin was found using biochemical methods by Fukuda in 2005. In this study, we examined the interaction between SNAP25-EGFP and mRFP-Rabphilin during the stimulated exocytosis within neuroendocrine PC12 cells through getting the FLIM images by the FLIM/FRET technique. The PC12 cells have P2X and P2Y receptors. If the receptors detected ATP, the calcium concentration in cells would be increased, and it would occur exocytosis. We found that during the usage of ATP for less than 10 seconds, the fluorescent lifetime distribution of samples which contain both GFP and RFP have shift about 170 ps. That is, during the short time when cells are stimulated and exocytosis occurs, FRET of SNAP25 and Rabphilin happens. Therefore, we believe that when exocytosis occurs, SNAP25 and Rabphilin would have interaction.