Visualization of the orai1 homodimer and the functional coupling of orai1-stim1 by live-cell fluorescence lifetime imaging

Ping Chun Huang, Tai Yu Chiu, Li Chun Wang, Hsiao Chuan Teng, Fu Jen Kao, De Ming Yang*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

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 Δ1- 90+Δ267-301 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.

Original languageEnglish
Pages (from-to)313-326
Number of pages14
JournalMicroscopy and Microanalysis
Volume16
Issue number3
DOIs
StatePublished - Jun 2010

Keywords

  • fluorescence lifetime imaging microscopy (FLIM)
  • fluorescence resonance energy transfer (FRET)
  • green fluorescent proteins (GFPs)
  • store-operated calcium channels (SOCs)
  • time-correlated single-photon counting (TCSPC)

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