X-ray-based living-cell motion analysis of individual serotonin receptors

Kazuhiro Mio, Masaki Ishihara, Shoko Fujimura, Daisuke Sasaki, Shunsuke Nozawa, Kohei Ichiyanagi, Ryo Fukaya, Shin ichi Adachi, Masahiro Kuramochi, Hiroshi Sekiguchi, Tai Kubo, Yuji C. Sasaki

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)

Abstract

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins, which transmit extracellular signals inside cells via activating G proteins. GPCRs are involved in a wide variety of physiological functions, such as signal sensing, immune system processes, and neurotransmission. Although the structures and functions of GPCRs have been well studied, little has been known about their real-time dynamics on live cells. In this study, we used Diffracted X-ray Tracking (DXT) and Diffracted X-ray Blinking (DXB) techniques for analysis. These methods are very precise single-molecular analytical techniques that elucidate protein dynamics by analyzing the diffraction spots from the gold nanocrystals labeled on the protein surface. DXT tracks diffraction spot movements, whereas DXB analyzes continuation of signals by calculating the autocorrelation function of each pixel from the recorded data. Serotonin receptor subtype 2A (5-HT2A receptors) were transiently expressed on HEK 293 cells, and the gold nanocrystals were attached to the N-terminally introduced FLAG-tag via anti-FLAG antibodies. Fast- and mid-range motions were recorded by DXT with 100μs and 1.25 ms/frame rate, respectively. Slow-range motion was obtained using the DXB method with 100 ms/frame rate. An agonist interestingly suppressed the fluctuations of 5-HT2A receptors at the microsecond-ranged fast measurement. On the contrary, the motion was enhanced by the agonist in the hundred-millisecond-ranged slow time scale. These dual-natured data may suggest that we succeeded in extracting different modes of receptor's motion on live cells; microsecond ranged fluctuation on the cell membrane, and millisecond-ranged dynamic movement comprising interactions with intracellular signaling molecules.

Original languageEnglish
Pages (from-to)306-313
Number of pages8
JournalBiochemical and biophysical research communications
Volume529
Issue number2
DOIs
Publication statusPublished - 2020 Aug 20
Externally publishedYes

Keywords

  • 5-HT
  • GPCR
  • Intramolecular dynamics
  • Live cell
  • X-ray diffraction analysis

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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