Visualizing the mechanical activation of Src

Abstract

The mechanical environment crucially influences many cell functions1. However, it remains largely mysterious how mechanical stimuli are transmitted into biochemical signals. Src is known to regulate the integrin–cytoskeleton interaction2, which is essential for the transduction of mechanical stimuli3,4,5. 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.

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Figure 1: The domain structure, schematic representation and in vitro characterizations of the Src reporter.
Figure 2: Characterization of the Src reporters.
Figure 3: Directional and long-range propagation of Src induced by mechanical force.
Figure 4: Actin filaments and microtubules are essential for the polarized and long-range Src activation.

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Acknowledgements

We thank J. Zhang and A. Y. Ting for their assistance and advice; J.-L. Guan, D. C. Flynn and J. Cooper for providing FAK protein, MEF cells, c-Yes plasmid and SYF cell lines. We also thank B. N. G. Giepmans and C. Hauser for their comments on the paper, and Q. Xiong for technical support. This work was supported by the National Institutes of Health (S.C., R.Y.T. and M.W.B.), the Howard Hughes Medical Institute (R.Y.T.), the Airforce Office of Scientific Research (M.W.B.), the Arnold and Mabel Beckman Foundation (E.L.B.) and the Alliance for Cellular Signaling Grant (R.Y.T., S.C. and Y.W.).

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Correspondence to Shu Chien.

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The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Materials

This file contains Supplementary Methods, Supplementary Figure Legends and Supplementary Movie Legends. (DOC 72 kb)

Supplementary Figures

This file contains Supplementary Figures S1-S7. (PPT 1203 kb)

Supplementary Movie S1

The EGF-induced FRET response in HeLa cells expressing the Src reporter. (MOV 207 kb)

Supplementary Movie S2

The effects of EGF stimulation and washout on FRET responses of the monomeric Src reporter in HeLa cells. (MOV 4949 kb)

Supplementary Movie S3

A fibronectin-coated bead induced local FRET responses in HUVECs. (MOV 3253 kb)

Supplementary Movie S4

PP1 reversed the local FRET response in HUVECs induced by fibronectin-coated beads. (MOV 2471 kb)

Supplementary Movie S5

The laser-tweezer-traction on a fibronectin-coated bead induced FRET responses of the cytosolic Src reporter in HUVECs. (MOV 4093 kb)

Supplementary Movie S6

The laser-tweezer-traction on a polylysine-coated bead did not induce FRET responses in HUVECs. (MOV 8073 kb)

Supplementary Movie S7

PP1 reversed the EGF-induced FRET responses of the membrane-targeted Src reporter in HeLa cells. (MOV 459 kb)

Supplementary Movie S8

Pretreatment with PP1 inhibited the EGF-induced FRET responses of the membrane-targeted Src reporter in HeLa cells. (MOV 1017 kb)

Supplementary Movie S9

The laser-tweezer-traction induced directional and long-range propagation of FRET responses of the membrane-targeted Src reporter in HUVECs. (MOV 6434 kb)

Supplementary Movie S10

The laser-tweezer traction on a fibronectin-coated bead did not induce FRET responses of the Y662F/Y664F mutant of the membrane-targeted Src reporter. (MOV 1759 kb)

Supplementary Movie S11

The laser-tweezer traction on a fibronectin-coated bead did not induce FRET responses of the R175V mutant of the membrane-targeted Src reporter. (MOV 1684 kb)

Supplementary Movie S12

A highlighted cell with a clear FRET wave propagation away from the stimulation site upon force application. (MOV 7469 kb)

Supplementary Movie S13

Cytochalasin D blocked the directional and long-range activation of Src in response to mechanical force. (MOV 6768 kb)

Supplementary Movie S14

Nocodazole blocked the directional and long-range activation of Src in response to mechanical force. (MOV 8909 kb)

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Wang, Y., Botvinick, E., Zhao, Y. et al. Visualizing the mechanical activation of Src. Nature 434, 1040–1045 (2005). https://doi.org/10.1038/nature03469

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