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Visualization of the Orai1 Homodimer and the Functional Coupling of Orai1-STIM1 by Live-Cell Fluorescence Lifetime Imaging

Published online by Cambridge University Press:  09 April 2010

Ping-Chun Huang
Affiliation:
Department of Medical Research and Education, Taipei Veterans General Hospital Taipei, Taiwan, Republic of China Institute of Biophotonics, School of Medical Technology and Engineering, National Yang-Ming University, Taipei, Taiwan, Republic of China
Tai-Yu Chiu
Affiliation:
Department of Medical Research and Education, Taipei Veterans General Hospital Taipei, Taiwan, Republic of China Institute of Biophotonics, School of Medical Technology and Engineering, National Yang-Ming University, Taipei, Taiwan, Republic of China
Li-Chun Wang
Affiliation:
Department of Medical Research and Education, Taipei Veterans General Hospital Taipei, Taiwan, Republic of China
Hsiao-Chuan Teng
Affiliation:
Department of Medical Research and Education, Taipei Veterans General Hospital Taipei, Taiwan, Republic of China
Fu-Jen Kao
Affiliation:
Institute of Biophotonics, School of Medical Technology and Engineering, National Yang-Ming University, Taipei, Taiwan, Republic of China
De-Ming Yang*
Affiliation:
Department of Medical Research and Education, Taipei Veterans General Hospital Taipei, Taiwan, Republic of China Institute of Biophotonics, School of Medical Technology and Engineering, National Yang-Ming University, Taipei, Taiwan, Republic of China
*
Corresponding author. E-mail: yang.deming@gmail.com
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Abstract

The Orai1-STIM1 constructed store-operated Ca2+ channels (SOCs) have been found to exert several essential Ca2+ entry/signaling cascades, e.g., the generation of immune response in T lymphocytes. Although biochemical and novel imaging evidence appear to indicate that Orai1 and STIM1 interact with each other to achieve store-operated Ca2+ entry (SOCE), the detailed mechanism of functional SOCE in situ has yet to be fully understood. In this study, green fluorescence protein (EGFP as donor) targeted to either the N- or C-terminal of Orai1 (wild type or 1-90+267-301 double deletion type) and mOrange (as acceptor) tagged STIM1 were used to comprise a fluorescence resonance energy transfer (FRET) pair within living PC12 cells. The fluorescence lifetime map and histogram/distribution of each single cell, determined by one-photon excitation fluorescence lifetime imaging microscopy (FLIM), was used to visualize FRET and show the Orai1 homodimer and Orai1-STIM1 binding. Both the color-coded lifetime map and the distribution of EGFP-tagged Orai1 significantly changed after the administration of thapsigargin, the SOCE stimulating agent. The FRET efficiency from each experimental set was also calculated and compared using double exponential analysis. In summary, we show the detailed interactions Orai1-Orai1 and Orai1-STIM1 within intact living cells by using the FLIM-FRET technique.

Type
Biological Applications
Copyright
Copyright © Microscopy Society of America 2010

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