title: Visualizing the mechanical activation of Src.
abstract: 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.
authors.affiliation: Department of Bioengineering and the Whitaker Institute of Biomedical Engineering, La Jolla, California 92093, USA.
authors.forename: Yingxiao
authors.initials: Y
authors.lastname: Wang
authors.forename: Elliot L
authors.initials: EL
authors.lastname: Botvinick
authors.forename: Yihua
authors.initials: Y
authors.lastname: Zhao
authors.forename: Michael W
authors.initials: MW
authors.lastname: Berns
authors.forename: Shunichi
authors.initials: S
authors.lastname: Usami
authors.forename: Roger Y
authors.initials: RY
authors.lastname: Tsien
authors.forename: Shu
authors.initials: S
authors.lastname: Chien
mesh_terms: 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
chemical_list: D005353:Fibronectins; D015335:Molecular Probes; D016392:Proto-Oncogene Proteins pp60(c-src)