β1- and αv-class integrins cooperate to regulate myosin II during rigidity sensing of fibronectin-based microenvironments.
TL;DR: This study assigns specific functions to distinct fibronectin-binding integrins, demonstrating that α5β1integrins accomplish force generation, whereas αv-class Integrins mediate the structural adaptations to forces, which cooperatively enable cells to sense the rigidity of fibronECTin-based microenvironments.
Abstract: How different integrins that bind to the same type of extracellular matrix protein mediate specific functions is unclear. We report the functional analysis of β1- and αv-class integrins expressed in pan-integrin-null fibroblasts seeded on fibronectin. Reconstitution with β1-class integrins promotes myosin-II-independent formation of small peripheral adhesions and cell protrusions, whereas expression of αv-class integrins induces the formation of large focal adhesions. Co-expression of both integrin classes leads to full myosin activation and traction-force development on stiff fibronectin-coated substrates, with αv-class integrins accumulating in adhesion areas exposed to high traction forces. Quantitative proteomics linked αv-class integrins to a GEF-H1-RhoA pathway coupled to the formin mDia1 but not myosin II, and α5β1 integrins to a RhoA-Rock-myosin II pathway. Our study assigns specific functions to distinct fibronectin-binding integrins, demonstrating that α5β1integrins accomplish force generation, whereas αv-class integrins mediate the structural adaptations to forces, which cooperatively enable cells to sense the rigidity of fibronectin-based microenvironments.
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TL;DR: Progress towards understanding the molecular, cellular and tissue-level effects that promote mechanical homeostasis has helped to identify key questions for future research.
Abstract: Soft connective tissues at steady state are dynamic; resident cells continually read environmental cues and respond to them to promote homeostasis, including maintenance of the mechanical properties of the extracellular matrix (ECM) that are fundamental to cellular and tissue health. The mechanosensing process involves assessment of the mechanics of the ECM by the cells through integrins and the actomyosin cytoskeleton, and is followed by a mechanoregulation process, which includes the deposition, rearrangement or removal of the ECM to maintain overall form and function. Progress towards understanding the molecular, cellular and tissue-level effects that promote mechanical homeostasis has helped to identify key questions for future research.
1,449 citations
Journal Article•
TL;DR: As a new kind of substrate of IIF, beta( 2)GP I transfectant can be used to detect anti-beta(2)GP-I antibodies and keep the immunofluorescent property of HEp-2 cells in IFANA test and can be use as substrate for routine IFANA detection.
Abstract: OBJECTIVE To establish an indirect immunofluorescent test so as to improve the sensitivity and specificity of examination of antibodies to beta(2)-glycoprotein METHODS Full-length beta(2)GP cDNA was obtained from human hepatocellular cancer cell line HepG2 by RT-PCR and cloned into the mammalian expression vector pEGFP-C1 The recombinant plasmid pEGFP-beta(2)GP was transfected into HEp-2 cells RT-PCR, immunoblotting (IBT), confocal fluorescence microscopy, and indirect immunofluorescent test (IIF) were used to confirm the expression, localization, and antigenicity of fusion protein of green fluorescent protein (GFP) Serum specimens from 19 patients suspected as with secondary antiphospholipid syndrome (APS), 1 patient diagnosed as with primary APS, and 10 normal persons were detected with IIF-IgG-beta(2)GP1, ELISA-IgG-ACL, and ELISA-IgG-beta(2)GP I simultaneously RESULTS (1) The HEp-beta(2)GP I cells thus obtained retained their ability of expression of beta(2)GP-I-GFP for more than ten generations This beta(2)GP-I-GFP showed the antigenicity of beta(2)GP-I with a characteristic feature (2) Seven of the 20 serum specimens from APS patients showed characteristic immunofluorescent pattern No serum specimen from normal persons showed immunofluorescent staining The comparison of results of the three methods showed that the concordance between IIF-IgG-beta(2)GP I and ELISA-IgG-beta(2)GP I was the most perfect (Kappa = 0886) (3) HEp-beta(2)GP I retained the immunofluorescent property of HEp-2 cell CONCLUSION As a new kind of substrate of IIF, beta(2)GP I transfectant can be used to detect anti-beta(2)GP-I antibodies Transfeted HEp-2 cells keep the immunofluorescent property of HEp-2 cells in IFANA test and can be used as substrate for routine IFANA detection
777 citations
TL;DR: Sun, Guo, and Fässler review the function and regulation of integrin-mediated mechanotransduction and discuss how its dysregulation impacts cancer progession.
Abstract: Cells can detect and react to the biophysical properties of the extracellular environment through integrin-based adhesion sites and adapt to the extracellular milieu in a process called mechanotransduction. At these adhesion sites, integrins connect the extracellular matrix (ECM) with the F-actin cytoskeleton and transduce mechanical forces generated by the actin retrograde flow and myosin II to the ECM through mechanosensitive focal adhesion proteins that are collectively termed the “molecular clutch.” The transmission of forces across integrin-based adhesions establishes a mechanical reciprocity between the viscoelasticity of the ECM and the cellular tension. During mechanotransduction, force allosterically alters the functions of mechanosensitive proteins within adhesions to elicit biochemical signals that regulate both rapid responses in cellular mechanics and long-term changes in gene expression. Integrin-mediated mechanotransduction plays important roles in development and tissue homeostasis, and its dysregulation is often associated with diseases.
667 citations
Cites background from "β1- and αv-class integrins cooperat..."
...For instance, β1 integrins can assemble adhesion complex without myosin-driven contractility and exhibit fast translocation in FAs, whereas β3 integrins are stationary in FAs and quickly internalized when force is released (Rossier et al., 2012; Schiller et al., 2013; Yu et al., 2015)....
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...It is also important to note that there is no simple correlation between the adhesion size and the magnitude of the transmitted force (Oakes et al., 2012; Schiller et al., 2013), Figure 1....
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...…also important to note that there is no simple correlation between the adhesion size and the magnitude of the transmitted force (Oakes et al., 2012; Schiller et al., 2013), Integrin-mediated mechanotransduction • Sun et al. 447 as small NAs may also transmit forces that are high enough to drive…...
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...Both RhoA downstream pathways are required for optimal cellular tension and rigidity sensing (Schiller et al., 2013)....
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TL;DR: An overview of how integrin function is regulated from both a biochemical and a mechanical perspective, affecting integrin cell-surface availability, binding properties, activation or clustering is provided, and how this biomechanical regulation allows integrins to respond to different ECM physicochemical properties and signals.
Abstract: Integrins, and integrin-mediated adhesions, have long been recognized to provide the main molecular link attaching cells to the extracellular matrix (ECM) and to serve as bidirectional hubs transmitting signals between cells and their environment. Recent evidence has shown that their combined biochemical and mechanical properties also allow integrins to sense, respond to and interact with ECM of differing properties with exquisite specificity. Here, we review this work first by providing an overview of how integrin function is regulated from both a biochemical and a mechanical perspective, affecting integrin cell-surface availability, binding properties, activation or clustering. Then, we address how this biomechanical regulation allows integrins to respond to different ECM physicochemical properties and signals, such as rigidity, composition and spatial distribution. Finally, we discuss the importance of this sensing for major cell functions by taking cell migration and cancer as examples.
652 citations
TL;DR: It is shown that in response to matrix rigidity and density, force transmission and transduction are explained by the mechanical properties of the actin–talin–integrin–fibronectin clutch.
Abstract: Cell function depends on tissue rigidity, which cells probe by applying and transmitting forces to their extracellular matrix, and then transducing them into biochemical signals. Here we show that in response to matrix rigidity and density, force transmission and transduction are explained by the mechanical properties of the actin-talin-integrin-fibronectin clutch. We demonstrate that force transmission is regulated by a dynamic clutch mechanism, which unveils its fundamental biphasic force/rigidity relationship on talin depletion. Force transduction is triggered by talin unfolding above a stiffness threshold. Below this threshold, integrins unbind and release force before talin can unfold. Above the threshold, talin unfolds and binds to vinculin, leading to adhesion growth and YAP nuclear translocation. Matrix density, myosin contractility, integrin ligation and talin mechanical stability differently and nonlinearly regulate both force transmission and the transduction threshold. In all cases, coupling of talin unfolding dynamics to a theoretical clutch model quantitatively predicts cell response.
543 citations
References
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TL;DR: By following this protocol, investigators are able to gain an in-depth understanding of the biological themes in lists of genes that are enriched in genome-scale studies.
Abstract: DAVID bioinformatics resources consists of an integrated biological knowledgebase and analytic tools aimed at systematically extracting biological meaning from large gene/protein lists. This protocol explains how to use DAVID, a high-throughput and integrated data-mining environment, to analyze gene lists derived from high-throughput genomic experiments. The procedure first requires uploading a gene list containing any number of common gene identifiers followed by analysis using one or more text and pathway-mining tools such as gene functional classification, functional annotation chart or clustering and functional annotation table. By following this protocol, investigators are able to gain an in-depth understanding of the biological themes in lists of genes that are enriched in genome-scale studies.
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Abstract: Efficient analysis of very large amounts of raw data for peptide identification and protein quantification is a principal challenge in mass spectrometry (MS)-based proteomics. Here we describe MaxQuant, an integrated suite of algorithms specifically developed for high-resolution, quantitative MS data. Using correlation analysis and graph theory, MaxQuant detects peaks, isotope clusters and stable amino acid isotope-labeled (SILAC) peptide pairs as three-dimensional objects in m/z, elution time and signal intensity space. By integrating multiple mass measurements and correcting for linear and nonlinear mass offsets, we achieve mass accuracy in the p.p.b. range, a sixfold increase over standard techniques. We increase the proportion of identified fragmentation spectra to 73% for SILAC peptide pairs via unambiguous assignment of isotope and missed-cleavage state and individual mass precision. MaxQuant automatically quantifies several hundred thousand peptides per SILAC-proteome experiment and allows statistically robust identification and quantification of >4,000 proteins in mammalian cell lysates.
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6,096 citations
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4,689 citations