Biphasic reinforcement of nascent adhesions by vinculin.

Autor: Baumann H; Department of Cell and Neurobiology, Zoological Institute, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany., Schwingel M; Department of Cell and Neurobiology, Zoological Institute, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany., Sestu M; Mechanisms of Cell Migration, Interdisciplinary Center for Clinical Research (IZKF), Faculty of Medicine, University of Leipzig, Leipzig, Germany., Burcza A; Department of Food Technology and Bioprocess Engineering, Max Rubner-Institut, Federal Research Institute of Nutrition and Food, Karlsruhe, Germany., Marg S; Mechanisms of Cell Migration, Interdisciplinary Center for Clinical Research (IZKF), Faculty of Medicine, University of Leipzig, Leipzig, Germany., Ziegler W; Department of Paediatric Kidney, Liver and Metabolic Diseases, Hannover Medical School, Hannover, Germany., Taubenberger AV; Center for Molecular and Cellular Bioengineering (CMCB), Technische Universität Dresden, Dresden, Germany., Muller DJ; Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland., Bastmeyer M; Department of Cell and Neurobiology, Zoological Institute, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.; Institute for Biological and Chemical Systems - Biological Information Processing (IBCS-BIP), Karlsruhe Institute of Technology (KIT), Eggenstein-Leopoldshafen, Germany., Franz CM; WPI Nano Life Science Institute, Kanazawa University, Kanazawa, Japan.
Jazyk: angličtina
Zdroj: Journal of molecular recognition : JMR [J Mol Recognit] 2023 Jun; Vol. 36 (6), pp. e3012. Date of Electronic Publication: 2023 Apr 10.
DOI: 10.1002/jmr.3012
Abstrakt: Vinculin is an integral component of integrin adhesions, where it functions as a molecular clutch coupling intracellular contraction to the extracellular matrix. Quantitating its contribution to the reinforcement of newly forming adhesions, however, requires ultrasensitive cell force assays covering short time and low force ranges. Here, we have combined atomic force microscopy-based single-cell force spectroscopy (SCFS) and optical tweezers force spectroscopy to investigate the role of vinculin in reinforcement of individual nascent adhesions during the first 5 min of cell contact with fibronectin or vitronectin. At minimal adhesion times (5-10 s), mouse embryonic fibroblast (MEF) wildtype (wt) and vinculin knock-out (vin (-/-) ) cells develop comparable adhesion forces on the scale of several individual integrin-ligand bonds, confirming that vinculin is dispensable for adhesion initiation. In contrast, after 60 to 120 s, adhesion strength and traction reinforce quickly in wt cells, while remaining low in vin (-/-) cells. Re-expression of full-length vinculin or a constitutively active vinculin mutant (vinT12) in MEF vin (-/-) cells restored adhesion and traction with the same efficiency, while vinculin with a mutated talin-binding head region (vinA50I) or missing the actin-binding tail-domain (vin880) was ineffective. Integrating total internal reflection fluorescence imaging into the SCFS setup furthermore enabled us to correlate vinculin-green fluorescent protein (GFP) recruitment to nascent adhesion sites with the built-up of vinculin-dependent adhesion forces directly. Vinculin recruitment and cell adhesion reinforcement followed synchronous biphasic patterns, suggesting vinculin recruitment, but not activation, as the rate-limiting step for adhesion reinforcement. Combining sensitive SCFS with fluorescence microscopy thus provides insight into the temporal sequence of vinculin-dependent mechanical reinforcement in nascent integrin adhesions.
(© 2023 John Wiley & Sons Ltd.)
Databáze: MEDLINE
Externí odkaz: