In-depth fluorescence lifetime imaging analysis revealing SNAP25A-rabphilin 3A interactions

Jiung De Lee, Ping Chun Huang, Yi Cheng Lin, Lung Sen Kao, Chien Chang Huang, Fu Jen Kao, Chung Chih Lin, De Ming Yang*


研究成果: Article同行評審

12 引文 斯高帕斯(Scopus)


The high sensitivity and spatial resolution enabled by two-photon excitation fluorescence lifetime imaging microscopy/fluorescence resonance energy transfer (2PE-FLIM/FRET) provide an effective approach that reveals protein-protein interactions in a single cell during stimulated exocytosis. Enhanced green fluorescence protein (EGFP)-labeled synaptosomal associated protein of 25 kDa (SNAP25A) and red fluorescence protein (mRFP)-labeled Rabphillin 3A (RPH3A) were co-expressed in PC12 cells as the FRET donor and acceptor, respectively. The FLIM images of EGFP-SNAP25A suggested that SNAP25A/RPH3A interaction was increased during exocytosis. In addition, the multidimensional (three-dimensional with time) nature of the 2PE-FLIM image datasets can also resolve the protein interactions in the z direction, and we have compared several image analysis methods to extract more accurate and detailed information from the FLIM images. Fluorescence lifetime was fitted by using one and two component analysis. The lifetime FRET efficiency was calculated by the peak lifetime (τpeak) and the left side of the half-peak width (τ1/2), respectively. The results show that FRET efficiency increased at cell surface, which suggests that SNAP25A/RPH3A interactions take place at cell surface during stimulated exocytosis. In summary, we have demonstrated that the 2PE-FLIM/FRET technique is a powerful tool to reveal dynamic SNAP25A/RPH3A interactions in single neuroendocrine cells.

頁(從 - 到)507-518
期刊Microscopy and Microanalysis
出版狀態Published - 12月 2008


深入研究「In-depth fluorescence lifetime imaging analysis revealing SNAP25A-rabphilin 3A interactions」主題。共同形成了獨特的指紋。