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

Fischer, L., Klingner, C., Schlichthärle, T., Strauss, M. T., Böttcher, R. T., Fässler, R., et al. (2021). Quantitative single-protein imaging reveals molecular complex formation of integrin, talin, and kindlin during cell adhesion. Nature Communications, 12(1): 919. doi:10.1038/s41467-021-21142-2.

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 Creators:
Fischer, Lisa1, Author              
Klingner, Christoph1, Author              
Schlichthärle, Thomas2, Author              
Strauss, Maximilian T.2, Author              
Böttcher, Ralph T.3, Author              
Fässler, Reinhard3, Author              
Jungmann, Ralf2, Author              
Grashoff, Carsten1, Author              
Affiliations:
1Grashoff, Carsten / Molecular Mechanotransduction, Max Planck Institute of Biochemistry, Max Planck Society, ou_1565150              
2Jungmann, Ralf / Molecular Imaging and Bionanotechnology, Max Planck Institute of Biochemistry, Max Planck Society, ou_2149679              
3Fässler, Reinhard / Molecular Medicine, Max Planck Institute of Biochemistry, Max Planck Society, ou_1565147              

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Free keywords: Science & Technology - Other Topics;
 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 beta 1-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. Single-molecule localisation microscopy is limited by low labeling and detection efficiencies of the molecular probes. Here the authors report a framework to obtain absolute molecular quantities on a true molecular scale; the data reveal a ternary adhesion complex underlying cell-matrix adhesion.

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Language(s): eng - English
 Dates: 2021
 Publication Status: Published online
 Pages: 10
 Publishing info: -
 Table of Contents: -
 Rev. Type: -
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Project name : DFG, GR3399/6-1 and Inst 211/867-1
Grant ID : -
Funding program : -
Funding organization : German Research Council
Project name : DFG JU 2957/1-1
Grant ID : -
Funding program : Emmy Noether Program
Funding organization : German Research Foundation
Project name : MolMap
Grant ID : 680241
Funding program : ERC Starting Grant
Funding organization : European Research Council

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Title: Nature Communications
  Abbreviation : Nat. Commun.
Source Genre: Journal
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Publ. Info: London : Nature Publishing Group
Pages: - Volume / Issue: 12 (1) Sequence Number: 919 Start / End Page: - Identifier: ISSN: 2041-1723
CoNE: https://pure.mpg.de/cone/journals/resource/2041-1723