Abstract
Contractile forces are the end effectors of cell migration, division, morphogenesis, wound healing and cancer invasion. Here we report optogenetic tools to upregulate and downregulate such forces with high spatiotemporal accuracy. The technology relies on controlling the subcellular activation of RhoA using the CRY2/CIBN light-gated dimerizer system. We fused the catalytic domain (DHPH domain) of the RhoA activator ARHGEF11 to CRY2-mCherry (optoGEF-RhoA) and engineered its binding partner CIBN to bind either to the plasma membrane or to the mitochondrial membrane. Translocation of optoGEF-RhoA to the plasma membrane causes a rapid and local increase in cellular traction, intercellular tension and tissue compaction. By contrast, translocation of optoGEF-RhoA to mitochondria results in opposite changes in these physical properties. Cellular changes in contractility are paralleled by modifications in the nuclear localization of the transcriptional regulator YAP, thus showing the ability of our approach to control mechanotransductory signalling pathways in time and space.
Highlights
Contractile forces are the end effectors of cell migration, division, morphogenesis, wound healing and cancer invasion
Further, that changes in cellular forces are paralleled by translocation of the transcriptional regulator YAP, indicating that our tools can be used to control mechanotransductory pathways
To control Rho-GEF localization we used the CRY2/CIBN light-gated dimerizer system. This system is based on two proteins, CRY2 and CIBN, which bind with high affinity upon exposure to blue light, but rapidly dissociate when illumination is switched off[30]
Summary
Contractile forces are the end effectors of cell migration, division, morphogenesis, wound healing and cancer invasion. The central role of contractile forces in cell function has motivated extensive research to identify the underlying molecular mechanisms and regulatory pathways From this fundamental knowledge several chemical compounds have been developed to tune cellular force generation. Small molecules and genetic perturbations are often used to target regulatory pathways, such as those controlling calcium levels or Rho GTPases[19] Despite their well-established effectiveness, the biochemical and genetic manipulations mentioned above are severely limited by their inability to provide tight spatiotemporal control of cell contractility. We report two optogenetic tools based on controlling the activity of endogenous RhoA to upregulate or downregulate cell contractility We show that these tools enable rapid, local and reversible changes in traction forces, cell–cell forces, and tissue compaction. Further, that changes in cellular forces are paralleled by translocation of the transcriptional regulator YAP, indicating that our tools can be used to control mechanotransductory pathways
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