Visualizing the mechanical activation of Src.
The mechanical environment crucially influences many cell functions. However, it remains largely mysterious how mechanical stimuli are transmitted into biochemical signals. Src is known to regulate the integrin-cytoskeleton interaction, which is essential for the transduction of mechanical stimuli. Using fluorescent resonance energy transfer (FRET), here we develop a genetically encoded Src reporter that enables the imaging and quantification of spatio-temporal activation of Src in live cells. We introduced a local mechanical stimulation to human umbilical vein endothelial cells (HUVECs) by applying laser-tweezer traction on fibronectin-coated beads adhering to the cells. Using the Src reporter, we observed a rapid distal Src activation and a slower directional wave propagation of Src activation along the plasma membrane. This wave propagated away from the stimulation site with a speed (mean +/- s.e.m.) of 18.1 +/- 1.7 nm s(-1). This force-induced directional and long-range activation of Src was abolished by the disruption of actin filaments or microtubules. Our reporter has thus made it possible to monitor mechanotransduction in live cells with spatio-temporal characterization. We find that the transmission of mechanically induced Src activation is a dynamic process that directs signals via the cytoskeleton to spatial destinations.
Wang
Yingxiao
Y
Department of Bioengineering and the Whitaker Institute of Biomedical Engineering, La Jolla, California 92093, USA.
Botvinick
Elliot L
EL
Zhao
Yihua
Y
Berns
Michael W
MW
Usami
Shunichi
S
Tsien
Roger Y
RY
Chien
Shu
S
D008841:Actin Cytoskeleton / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001696:Biomechanical Phenomena; D002448:Cell Adhesion; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D004789:Enzyme Activation; D005347:Fibroblasts; D005353:Fibronectins / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006801:Humans; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D051379:Mice; D008863:Microspheres; D008870:Microtubules / Q000378:metabolism; D008958:Models, Molecular; D015335:Molecular Probes / Q000032:analysis; D008969:Molecular Sequence Data; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics / Q000378:metabolism*; D014471:Umbilical Veins / Q000166:cytology
D005353:Fibronectins; D015335:Molecular Probes; D016392:Proto-Oncogene Proteins pp60(c-src)