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Kindlin3 regulates biophysical properties and mechanics of membrane to cortex attachment

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Abstract

Kindlin3 (K3), a FERM domain containing protein expressed in hematopoietic cells controls integrin activation and thus hemostatic and inflammatory responses. However, its role in the mechanics of plasma membrane remains unclear. Here, we show that genetic knockout of K3 in microglia and macrophages resulted in defective plasma membrane tension and membrane blebbing. Atomic force microscopy (AFM) of K3-deficient cells revealed a significant loss in membrane-to-cortex attachment (MCA), and consequently reduced membrane tension. This loss in MCA is amplified by the mislocalization of the cell cortex proteins—ezrin, radixin, and moesin (ERM)—to the plasma membrane of microglia and macrophages. Re-expression of K3 in K3-deficient macrophages rescued the defects and localization of ERMs implying a key role for K3 in MCA. Analysis of two K3 mutants, K3int affecting integrin binding and activation, and K3pxn/act disrupting binding to paxillin and actin but not integrin functions, demonstrated that the role of K3 in membrane mechanics is separate from integrin activation. The K3pxn/act mutant substantially diminished both membrane tension and Yes-associated protein (YAP) translocation to the nucleus, while preserving integrin activation, cell spreading, and migration. Together, our results show that K3 coordinates membrane mechanics, ERM protein recruitment to the membrane, and YAP translocation by linking integrin at the membrane to paxillin and actin of the cytoskeleton. This novel function of K3 is distinct from its role in integrin activation.

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All data generated or analyzed during this study are included in this published article (and its supplementary information files).

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Conflict of interests

The authors declare that they have no conflict of interests.

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Funding

This work was supported by grants from NIH; R01 HL071625 to T.V.B. and funds from National Science Foundation (CBET, Award # 1337859) to C.R.K. We acknowledge the use of the Cleveland Clinic Imaging Core equipment and services supported by NIH SIG grant 1S10RR026820-01.

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Authors and Affiliations

  1. Lerner Research Institute, Cleveland Clinic, Cleveland, OH, 44195, USA

    Tejasvi Dudiki, Huan Liu, Irina Zhevlakova, Chase Bertagnolli, Daniel W. Nascimento & Tatiana V. Byzova

  2. Chemical and Biomedical Engineering Department, Washkewicz College of Engineering, Cleveland State University, Cleveland, OH, 44115, USA

    Gautam Mahajan & Chandrasekhar R. Kothapalli

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  1. Tejasvi Dudiki
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  2. Gautam Mahajan
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Contributions

TD designed and performed experiments, performed data analysis, and wrote the manuscript. GM, HL, IZ, CB, and DN performed experiments and data analysis. CRK and TVB conceived experiments, analyzed data, wrote the manuscript, and secured funding.

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Correspondence to Chandrasekhar R. Kothapalli or Tatiana V. Byzova.

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Animal experimental procedures were performed in accordance with National Institutes of Health (NIH) guidelines on animal care and all protocols were approved by the Institutional Animal Care and Use Committee at Cleveland Clinic. The lentivirus infection was performed in accordance with the Cleveland Clinic IBC protocol.

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Dudiki, T., Mahajan, G., Liu, H. et al. Kindlin3 regulates biophysical properties and mechanics of membrane to cortex attachment. Cell. Mol. Life Sci. 78, 4003–4018 (2021). https://doi.org/10.1007/s00018-021-03817-7

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