Focal adhesions are controlled by microtubules through local contractility regulation

iLID system-based optogenetic construct of KANK1 protein (OptoKANK) was created for this study. It was made of (i) KANK1 talin binding domain (KN) fused with mApple at its N-terminus and LOV2ssrA at the C-terminus and (ii) remaining part of KANK1 molecule (ΔKN) fused with SSpB at the N-terminus and mEmerald at the C-terminus. Over-expression of OptoKANK was sufficient to displace endogenous KANK1 and thereby disconnect microtubules from the focal adhesions. Also, blue light illumination of focal adhesions visualized by vinculin-mIFP resulted in the sliding and disassembly in exactly similar manner as in wild-type cells containing endogenous KANK1 and KANK2. Thus, the effect of activation of OptoKANK is not the result of its interaction with endogenous KANK1/2.

Comparison of the dynamics of myosin filaments accumulation with those of microtubule and vinculin density in focal adhesion area showed that the appearance of myosin filaments at the proximal end of focal adhesion coincided with withdrawal of microtubules that were attracted to focal adhesions shortly after the onset of illumination. In turn, the moment when the density of myosin filaments approached the maximum preceded the process of focal adhesion disassembly.

Measurements of traction force at the focal adhesion area (approximately 4 pillars per focal adhesion) over the time revealed a significant increase of traction force upon illumination-induced OptoKANK activation compared to the non-illuminated area.

Accumulation of myosin filaments in proximity to focal adhesions and development of traction forces appeared to be critically important for the disassembly of focal adhesions upon forced targeting of microtubules by OptoKANK activation. Thus, targeting of microtubules to focal adhesions by OptoKANK activation results in transient accumulation of myosin-II filaments near the proximal end of the focal adhesions and bursts of traction force, which are required for focal adhesion disassembly.

It was found that in GEF-H1 depleted cells activation of OptoKANK by the illumination of focal adhesions did not result in increase of traction force applied to the focal adhesions (Fig. 1A). Consistently, the OptoKANK activation in GEF-H1 depleted cells did not trigger the sliding and disassembly of the focal adhesions.

Fig. 1: Rho activation by GEF-H1 is required for focal adhesion disassembly upon microtubule targeting.

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^1,2Mechanobiology Institute, National University of Singapore, Singapore. ³Grenoble Institute of Neuroscience, University Grenoble, France. ⁴University Grenoble Alpes, Institute for Advanced Biosciences, France. ⁵Department of Biomedical Engineering, National University of Singapore, Singapore. ⁶Courant Institute and Department of Biology, New York University, USA. ⁷Mechanobiology Institute, National University of Singapore, Singapore. ⁸Department of Molecular Biology, Weizmann Institute of Science, Israel.