TY - JOUR
T1 - X-ray-based living-cell motion analysis of individual serotonin receptors
AU - Mio, Kazuhiro
AU - Ishihara, Masaki
AU - Fujimura, Shoko
AU - Sasaki, Daisuke
AU - Nozawa, Shunsuke
AU - Ichiyanagi, Kohei
AU - Fukaya, Ryo
AU - Adachi, Shin ichi
AU - Kuramochi, Masahiro
AU - Sekiguchi, Hiroshi
AU - Kubo, Tai
AU - Sasaki, Yuji C.
N1 - Funding Information:
This work was supported by JSPS KAKENHI Grant Numbers JP17H05539 , JP18K06601 , JP26102748 and by JST CREST Grant Number JP18071859 , Japan. DXT and DXB experiments were performed with the approval of the Japan Synchrotron Radiation Research Institute (Proposal Nos 2017A1140, 2018A1417 and 2019A1498), and of the Photon Factory Program Advisory Committee (Proposal No. 2017G556).
Funding Information:
This work was supported by JSPS KAKENHI Grant Numbers JP17H05539, JP18K06601, JP26102748 and by JST CREST Grant Number JP18071859, Japan. DXT and DXB experiments were performed with the approval of the Japan Synchrotron Radiation Research Institute (Proposal Nos 2017A1140, 2018A1417 and 2019A1498), and of the Photon Factory Program Advisory Committee (Proposal No. 2017G556).
Publisher Copyright:
© 2020 Elsevier Inc.
PY - 2020/8/20
Y1 - 2020/8/20
N2 - 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.
AB - 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.
KW - 5-HT
KW - GPCR
KW - Intramolecular dynamics
KW - Live cell
KW - X-ray diffraction analysis
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U2 - 10.1016/j.bbrc.2020.05.200
DO - 10.1016/j.bbrc.2020.05.200
M3 - Article
C2 - 32703428
AN - SCOPUS:85087126416
VL - 529
SP - 306
EP - 313
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 2
ER -