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Quantitative single-protein imaging reveals molecular complex formation of integrin, talin, and kindlin during cell adhesion

Author

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  • Lisa S. Fischer

    (University of Münster
    Max Planck Institute of Biochemistry)

  • Christoph Klingner

    (University of Münster
    Max Planck Institute of Biochemistry)

  • Thomas Schlichthaerle

    (LMU Munich
    Max Planck Institute of Biochemistry)

  • Maximilian T. Strauss

    (LMU Munich
    Max Planck Institute of Biochemistry
    Max Planck Institute of Biochemistry)

  • Ralph Böttcher

    (Max Planck Institute of Biochemistry)

  • Reinhard Fässler

    (Max Planck Institute of Biochemistry)

  • Ralf Jungmann

    (LMU Munich
    Max Planck Institute of Biochemistry)

  • Carsten Grashoff

    (University of Münster
    Max Planck Institute of Biochemistry)

Abstract

Single-molecule localization microscopy (SMLM) enabling the investigation of individual proteins on molecular scales has revolutionized how biological processes are analysed in cells. However, a major limitation of imaging techniques reaching single-protein resolution is the incomplete and often unknown labeling and detection efficiency of the utilized molecular probes. As a result, fundamental processes such as complex formation of distinct molecular species cannot be reliably quantified. Here, we establish a super-resolution microscopy framework, called quantitative single-molecule colocalization analysis (qSMCL), which permits the identification of absolute molecular quantities and thus the investigation of molecular-scale processes inside cells. The method combines multiplexed single-protein resolution imaging, automated cluster detection, in silico data simulation procedures, and widely applicable experimental controls to determine absolute fractions and spatial coordinates of interacting species on a true molecular level, even in highly crowded subcellular structures. The first application of this framework allowed the identification of a long-sought ternary adhesion complex—consisting of talin, kindlin and active β1-integrin—that specifically forms in cell-matrix adhesion sites. Together, the experiments demonstrate that qSMCL allows an absolute quantification of multiplexed SMLM data and thus should be useful for investigating molecular mechanisms underlying numerous processes in cells.

Suggested Citation

  • Lisa S. Fischer & Christoph Klingner & Thomas Schlichthaerle & Maximilian T. Strauss & Ralph Böttcher & Reinhard Fässler & Ralf Jungmann & Carsten Grashoff, 2021. "Quantitative single-protein imaging reveals molecular complex formation of integrin, talin, and kindlin during cell adhesion," Nature Communications, Nature, vol. 12(1), pages 1-10, December.
  • Handle: RePEc:nat:natcom:v:12:y:2021:i:1:d:10.1038_s41467-021-21142-2
    DOI: 10.1038/s41467-021-21142-2
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    Cited by:

    1. Fan Lu & Liang Zhu & Thomas Bromberger & Jun Yang & Qiannan Yang & Jianmin Liu & Edward F. Plow & Markus Moser & Jun Qin, 2022. "Mechanism of integrin activation by talin and its cooperation with kindlin," Nature Communications, Nature, vol. 13(1), pages 1-19, December.
    2. Alexey Ferapontov & Marjan Omer & Isabelle Baudrexel & Jesper Sejrup Nielsen & Daniel Miotto Dupont & Kristian Juul-Madsen & Philipp Steen & Alexandra S. Eklund & Steffen Thiel & Thomas Vorup-Jensen &, 2023. "Antigen footprint governs activation of the B cell receptor," Nature Communications, Nature, vol. 14(1), pages 1-20, December.
    3. Mohamadreza Fazel & Michael J. Wester & David J. Schodt & Sebastian Restrepo Cruz & Sebastian Strauss & Florian Schueder & Thomas Schlichthaerle & Jennifer M. Gillette & Diane S. Lidke & Bernd Rieger , 2022. "High-precision estimation of emitter positions using Bayesian grouping of localizations," Nature Communications, Nature, vol. 13(1), pages 1-11, December.

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