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In-Depth Fluorescence Lifetime Imaging Analysis Revealing SNAP25A-Rabphilin 3A Interactions

Published online by Cambridge University Press:  06 November 2008

Jiung-De Lee ,
Ping-Chun Huang ,
Yi-Cheng Lin ,
Lung-Sen Kao ,
Chien-Chang Huang ,
Fu-Jen Kao ,
Chung-Chih Lin  and
De-Ming Yang
Jiung-De Lee
Affiliation:
Department of Medical Research and Education, Taipei Veterans General Hospital, National Yang-Ming University, Taipei 11217, Taiwan, Republic of China Institute of Biophotonics, School of Medical Technology and Engineering, National Yang-Ming University, Taipei 11217, Taiwan, Republic of China
Ping-Chun Huang
Affiliation:
Institute of Biophotonics, School of Medical Technology and Engineering, National Yang-Ming University, Taipei 11217, Taiwan, Republic of China
Yi-Cheng Lin
Affiliation:
Institute of Biophotonics, School of Medical Technology and Engineering, National Yang-Ming University, Taipei 11217, Taiwan, Republic of China
Lung-Sen Kao
Affiliation:
Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan, Republic of China
Chien-Chang Huang
Affiliation:
Graduate Institute of Biochemistry, School of Life Science, National Yang-Ming University, Taipei, Taiwan, Republic of China
Fu-Jen Kao
Affiliation:
Institute of Biophotonics, School of Medical Technology and Engineering, National Yang-Ming University, Taipei 11217, Taiwan, Republic of China
Chung-Chih Lin
Affiliation:
Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan, Republic of China
De-Ming Yang*
Affiliation:
Department of Medical Research and Education, Taipei Veterans General Hospital, National Yang-Ming University, Taipei 11217, Taiwan, Republic of China Institute of Biophotonics, School of Medical Technology and Engineering, National Yang-Ming University, Taipei 11217, Taiwan, Republic of China
*
Corresponding author. E-mail: yang.deming@gmail.com
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Abstract

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.

Keywords

two-photon excitation fluorescence microscopyfluorescence lifetime imaging microscopygreen fluorescent proteinexocytosisSNAP25ARabphilin 3A (RPH3A)fluorescence resonance energy transferimage analysis
Type
Multiphoton Microscopy–Special Section
Information
Microscopy and Microanalysis , Volume 14 , Issue 6 , December 2008 , pp. 507 - 518
Copyright
Copyright © Microscopy Society of America 2008

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