In-depth fluorescence lifetime imaging analysis revealing SNAP25A-Rabphilin 3A interactions.
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 (taupeak) and the left side of the half-peak width (tau1/2), 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.
Lee
Jiung-De
JD
Department of Medical Research and Education, Taipei Veterans General Hospital, National Yang-Ming University, Taipei 11217, Taiwan, Republic of China.
Huang
Ping-Chun
PC
Lin
Yi-Cheng
YC
Kao
Lung-Sen
LS
Huang
Chien-Chang
CC
Kao
Fu-Jen
FJ
Lin
Chung-Chih
CC
Yang
De-Ming
DM
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D005089:Exocytosis; D031541:Fluorescence Resonance Energy Transfer; D007091:Image Processing, Computer-Assisted; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D036641:Microscopy, Fluorescence, Multiphoton; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D055099:Neuroendocrine Cells / Q000166:cytology / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*; D000097624:Rabphilin-3A
D048868:Adaptor Proteins, Signal Transducing; D008164:Luminescent Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D000097624:Rabphilin-3A; C494210:Snap25 protein, rat