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

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 unk...

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Autores principales: Fischer, Lisa S., Klingner, Christoph, Schlichthaerle, Thomas, Strauss, Maximilian T., Böttcher, Ralph, Fässler, Reinhard, Jungmann, Ralf, Grashoff, Carsten
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7876120/
https://www.ncbi.nlm.nih.gov/pubmed/33568673
http://dx.doi.org/10.1038/s41467-021-21142-2
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author Fischer, Lisa S.
Klingner, Christoph
Schlichthaerle, Thomas
Strauss, Maximilian T.
Böttcher, Ralph
Fässler, Reinhard
Jungmann, Ralf
Grashoff, Carsten
author_facet Fischer, Lisa S.
Klingner, Christoph
Schlichthaerle, Thomas
Strauss, Maximilian T.
Böttcher, Ralph
Fässler, Reinhard
Jungmann, Ralf
Grashoff, Carsten
author_sort Fischer, Lisa S.
collection PubMed
description 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.
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spelling pubmed-78761202021-02-24 Quantitative single-protein imaging reveals molecular complex formation of integrin, talin, and kindlin during cell adhesion Fischer, Lisa S. Klingner, Christoph Schlichthaerle, Thomas Strauss, Maximilian T. Böttcher, Ralph Fässler, Reinhard Jungmann, Ralf Grashoff, Carsten Nat Commun Article 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. Nature Publishing Group UK 2021-02-10 /pmc/articles/PMC7876120/ /pubmed/33568673 http://dx.doi.org/10.1038/s41467-021-21142-2 Text en © The Author(s) 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Fischer, Lisa S.
Klingner, Christoph
Schlichthaerle, Thomas
Strauss, Maximilian T.
Böttcher, Ralph
Fässler, Reinhard
Jungmann, Ralf
Grashoff, Carsten
Quantitative single-protein imaging reveals molecular complex formation of integrin, talin, and kindlin during cell adhesion
title Quantitative single-protein imaging reveals molecular complex formation of integrin, talin, and kindlin during cell adhesion
title_full Quantitative single-protein imaging reveals molecular complex formation of integrin, talin, and kindlin during cell adhesion
title_fullStr Quantitative single-protein imaging reveals molecular complex formation of integrin, talin, and kindlin during cell adhesion
title_full_unstemmed Quantitative single-protein imaging reveals molecular complex formation of integrin, talin, and kindlin during cell adhesion
title_short Quantitative single-protein imaging reveals molecular complex formation of integrin, talin, and kindlin during cell adhesion
title_sort quantitative single-protein imaging reveals molecular complex formation of integrin, talin, and kindlin during cell adhesion
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7876120/
https://www.ncbi.nlm.nih.gov/pubmed/33568673
http://dx.doi.org/10.1038/s41467-021-21142-2
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