TY - JOUR
T1 - Visualizing Spatiotemporal Dynamics of Intercellular Mechanotransmission upon Wounding
AU - Wang, Pengzhi
AU - Liang, Jing
AU - Shi, Linda Z.
AU - Wang, Yi
AU - Zhang, Ping
AU - Ouyang, Mingxing
AU - Preece, Daryl
AU - Peng, Qin
AU - Shao, Lunan
AU - Fan, Jason
AU - Sun, Jie
AU - Li, Shawn S.
AU - Berns, Michael W.
AU - Zhao, Huimin
AU - Wang, Yingxiao
N1 - Funding Information:
The authors thank Drs. Alice Y. Ting and Michiyuki Matsuda for valuable constructs. The authors are grateful to the members of Dr. Michael W. Berns, Dr. Huimin Zhao, and Dr. Yingxiao Wang’s laboratories for their helpful assistance and input. This work is supported, in part, by Grants from NIH HL121365, GM125379, CA204704, and CA209629 (Y.W.), NSF CBET1360341 (to Y.W.), and by Grants from AFOSR FA9550-08-1-0284, AFOSR FA9550-17-1-0193, and gifts from the Hoag Family Foundation, Huntington Beach CA, the David and Lucille Packard Foundation, Los Altos, CA, and the Beckman Laser Institute Foundation (to M.W.B.). The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Funding Information:
The authors thank Drs. Alice Y. Ting and Michiyuki Matsuda for valuable constructs. The authors are grateful to the members of Dr. Michael W. Berns, Dr. Huimin Zhao, and Dr. Yingxiao Wang's laboratories for their helpful assistance and input. This work is supported, in part, by Grants from NIH HL121365, GM125379, CA204704, and CA209629 (Y.W.), NSF CBET1360341 (to Y.W.), and by Grants from AFOSR FA9550-08-1-0284, AFOSR FA9550-17-1-0193, and gifts from the Hoag Family Foundation, Huntington Beach CA, the David and Lucille Packard Foundation, Los Altos, CA, and the Beckman Laser Institute Foundation (to M.W.B.). The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Publisher Copyright:
© 2018 American Chemical Society.
PY - 2018/9/19
Y1 - 2018/9/19
N2 - During cell-to-cell communications, the interplay between physical and biochemical cues is essential for informational exchange and functional coordination, especially in multicellular organisms. However, it remains a challenge to visualize intercellular signaling dynamics in single live cells. Here, we report a photonic approach, based on laser microscissors and Förster resonance energy transfer (FRET) microscopy, to study intercellular signaling transmission. First, using our high-throughput screening platform, we developed a highly sensitive FRET-based biosensor (SCAGE) for Src kinase, a key regulator of intercellular interactions and signaling cascades. Notably, SCAGE showed a more than 40-fold sensitivity enhancement than the original biosensor in live mammalian cells. Next, upon local severance of physical intercellular connections by femtosecond laser pulses, SCAGE enabled the visualization of a transient Src activation across neighboring cells. Lastly, we found that this observed transient Src activation following the loss of cell-cell contacts depends on the passive structural support of cytoskeleton but not on the active actomyosin contractility. Hence, by precisely introducing local physical perturbations and directly visualizing spatiotemporal transmission of ensuing signaling events, our integrated approach could be broadly applied to mimic and investigate the wounding process at single-cell resolutions. This integrated approach with highly sensitive FRET-based biosensors provides a unique system to advance our in-depth understanding of molecular mechanisms underlying the physical-biochemical basis of intercellular coupling and wounding processes.
AB - During cell-to-cell communications, the interplay between physical and biochemical cues is essential for informational exchange and functional coordination, especially in multicellular organisms. However, it remains a challenge to visualize intercellular signaling dynamics in single live cells. Here, we report a photonic approach, based on laser microscissors and Förster resonance energy transfer (FRET) microscopy, to study intercellular signaling transmission. First, using our high-throughput screening platform, we developed a highly sensitive FRET-based biosensor (SCAGE) for Src kinase, a key regulator of intercellular interactions and signaling cascades. Notably, SCAGE showed a more than 40-fold sensitivity enhancement than the original biosensor in live mammalian cells. Next, upon local severance of physical intercellular connections by femtosecond laser pulses, SCAGE enabled the visualization of a transient Src activation across neighboring cells. Lastly, we found that this observed transient Src activation following the loss of cell-cell contacts depends on the passive structural support of cytoskeleton but not on the active actomyosin contractility. Hence, by precisely introducing local physical perturbations and directly visualizing spatiotemporal transmission of ensuing signaling events, our integrated approach could be broadly applied to mimic and investigate the wounding process at single-cell resolutions. This integrated approach with highly sensitive FRET-based biosensors provides a unique system to advance our in-depth understanding of molecular mechanisms underlying the physical-biochemical basis of intercellular coupling and wounding processes.
KW - FRET imaging
KW - active actomyosin contractility
KW - directed evolution
KW - high-throughput screening
KW - highly sensitive FRET-based biosensor SCAGE
KW - laser-induced wounding
KW - passive structural support of cytoskeleton
KW - transient Src activation
UR - http://www.scopus.com/inward/record.url?scp=85052992015&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85052992015&partnerID=8YFLogxK
U2 - 10.1021/acsphotonics.8b00383
DO - 10.1021/acsphotonics.8b00383
M3 - Article
AN - SCOPUS:85052992015
VL - 5
SP - 3565
EP - 3574
JO - ACS Photonics
JF - ACS Photonics
SN - 2330-4022
IS - 9
ER -