The interrelationship between microtubules and the actin cytoskeleton in mechanoregulation of integrin-mediated adhesions is poorly understood. Here, we show that the effects of microtubules on two major types of cell-matrix adhesion, focal adhesions and podosomes, are mediated by KANK family proteins connecting the adhesion protein talin with microtubule tips. Both total microtubule disruption and microtubule uncoupling from adhesions by manipulations with KANKs trigger a massive assembly of myosin IIA filaments, augmenting focal adhesions and disrupting podosomes. Myosin IIA filaments are indispensable effectors in the microtubule-driven regulation of integrin-mediated adhesions. Myosin IIA filament assembly depends on Rho activation by the RhoGEF GEF-H1, which is trapped by microtubules when they are connected with integrin-mediated adhesions via KANK proteins but released after their disconnection. Thus, microtubule capture by integrin-mediated adhesions modulates the GEF-H1-dependent effect of microtubules on the assembly of myosin IIA filaments. Subsequent actomyosin reorganization then remodels the focal adhesions and podosomes, closing the regulatory loop.
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All data generated or analysed during this study are included in this published article (and its Supplementary Information files). Raw datasets generated during and/or analysed during the current study are available from the corresponding authors on reasonable request.
Custom-written code used to analyse the data in the current study is available from the corresponding authors on reasonable request.
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We thank A. Akhmanova (Utrecht University, Netherlands), R. Fäasler (Max Plank Institute for Biochemistry, Martinsried, Germany), C. M. Waterman (National Institutes of Health, USA), R. Zaidel-Bar (Mechanobiology Institute, Singapore) and M. Dodding for providing constructs used in this study. We are grateful to A. Akhmanova for useful discussions and constructive criticism. We thank D. Pitta de Araujo (MBI Science Communications Unit) for help with Fig. 6. This research is supported by the National Research Foundation, Prime Minister’s Office, Singapore, and the Ministry of Education under the Research Centres of Excellence programme through the Mechanobiology Institute, Singapore (ref no. R-714-006-006-271) (A.D.B., N.B.M.R., V.T. and M.N.) and Singapore Ministry of Education Academic Research Fund Tier 3 MOE grant no. MOE2016-T3-1-002 (A.D.B., Y.N.). A.D.B. also acknowledges support from a Maimonides Israeli–France grant (Israeli Ministry of Science Technology and Space) and EU Marie Skłodowska-Curie Network InCeM (project ID 642866) at the Weizmann Institute of Science. N.B.M.R. is also funded by a joint National University of Singapore–King’s College London graduate studentship. G.E.J. is supported by the Medical Research Council, UK (G1100041, MR/K015664) and the generous provision of a visiting professorship from the Mechanobiology Institute, Singapore. P.K. and Z.Z. are funded by the Ministry of Education Academic Research Fund Tier 2 (MOE-T2-1-124), the Mechanobiology Institute seed funding, the National Research Foundation Fellowship (NRF-NRFF-2011-04) and the National Research Foundation Competitive Research Programme (NRF2012NRF-CRP001-084).
Supplementary Notes, Supplementary Figs. 1–13, Supplementary Table 1, Supplementary Video Legends 1–17, Supplementary Refs. 1–39
The microtubule plus ends of an HT1080 cell transfected with control siRNA are accumulated at focal adhesions concentrated within adhesive islands
The microtubule plus ends were randomly distributed over adhesive islands and spaces between them in an HT1080 cell depleted of KANK1 and KANK2
Microtubule outgrowth induced transient disassembly of focal adhesions in an HT1080 cell
Rapamycin-induced linking of two parts of the KANK1 molecule triggered disassembly of focal adhesions in an HT1080 cell
Microtubule disruption triggered massive assembly of myosin II filaments and disassembly of podosomes in a THP-1 cell
Microtubule disruption triggered massive assembly of myosin II filaments and augmentation of focal adhesion in an HT1080 cell
Microtubule outgrowth induced disassembly of myosin II filaments in an HT1080 cell
Transient decrease in traction forces after microtubule outgrowth in an HT1080 cell
KANK2 knockdown suppressed disassembly of myosin II filaments induced by microtubule outgrowth in an HT1080 cell
Activation of RhoA by CN03 augmented myosin II filament formation and podosome disruption in a THP-1 cell
Dynamics of podosomes and myosin II filaments in a control THP-1 cell
ROCK inhibition by Y-27632 did not affect podosome dynamics in a THP-1 cell
Inhibition of ROCK by Y-27632 disrupted myosin II filaments and prevented nocodazole-induced disassembly of podosomes in a THP-1 cell
Disruption of myosin II filaments by Y-27632 resulted in recovery of podosomes in a THP-1 cell pretreated with nocodazole
Disruption of myosin II filaments by Y-27632 resulted in recovery of podosomes in a KANK1-depleted THP-1 cell
Constitutively active RhoA and ROCK prevented the disruption of focal adhesions on microtubule outgrowth in HT1080 cells
GEF-H1 knockdown suppressed disruption of myosin II filaments and prevented disassembly of focal adhesions on microtubule outgrowth in an HT1080 cell