33 research outputs found
Proteomic analysis of integrin-associated complexes from mesenchymal stem cells
PURPOSE: Multipotent mesenchymal stem cells (MSCs) have the capability to differentiate down adipocyte, osteocyte and chondrocyte lineages and as such offer a range of potential therapeutic applications. The composition and stiffness of the extracellular matrix (ECM) environment that surrounds cells dictates their transcriptional programme, thereby affecting stem cell lineage decisionâmaking. Cells sense force via linkages between themselves and their microenvironment, and this is transmitted by integrin receptors and associated adhesion signalling complexes. To identify regulators of MSC force sensing, we sought to catalogue MSC integrinâassociated adhesion complex composition. EXPERIMENTAL DESIGN: Adhesion complexes formed by MSCs plated on the ECM ligand fibronectin were isolated and characterised by MS. Identified proteins were interrogated by comparison to a literatureâbased reference set of cell adhesionârelated components and using ontological and proteinâprotein interaction network analyses. RESULTS: Adhesion complexâspecific proteins in MSCs were identified that comprised predominantly cell adhesionârelated adaptors and actin cytoskeleton regulators. Furthermore, LIM domainâcontaining proteins in MSC adhesion complexes were highlighted, which may act as forceâsensing components. CONCLUSION AND CLINICAL RELEVANCE: These data provide a valuable resource of information regarding the molecular connections that link integrins and adhesion signalling in MSCs, and as such may present novel opportunities for therapeutic intervention
Osteoblast mineralization requires ÎČ1 integrin/ICAP-1âdependent fibronectin deposition
ICAP-1 prevents recruitment of kindlin-2 to ÎČ1 integrin to control dynamics of fibrillar adhesion sites, fibronectin deposition, and osteoblast mineralization during bone formation
Definition of a consensus integrin adhesome and its dynamics during adhesion complex assembly and disassembly
Integrin receptor activation initiates the formation of integrin adhesion complexes (IACs) at the cell membrane that transduce adhesion-dependent signals to control a multitude of cellular functions. Proteomic analyses of isolated IACs have revealed an unanticipated molecular complexity; however, a global view of the consensus composition and dynamics of IACs is currently lacking. Here, we have integrated several IAC proteomes and generated a 2,412-protein integrin adhesome. Analysis of this dataset reveals the functional diversity of proteins in IACs and establishes a consensus adhesome of 60 proteins. The consensus adhesome likely represents a core cell adhesion machinery, centred around four axes comprising ILK-PINCH-kindlin, FAK-paxillin, talin-vinculin and α-actinin-zyxin-VASP, and includes underappreciated IAC components such as Rsu-1 and caldesmon. Proteomic quantification of IAC assembly and disassembly detailed the compositional dynamics of the core cell adhesion machinery. The definition of this consensus view of integrin adhesome components provides a resource for the research community
Microtubule-dependent modulation of adhesion complex composition.
The microtubule network regulates the turnover of integrin-containing adhesion complexes to stimulate cell migration. Disruption of the microtubule network results in an enlargement of adhesion complex size due to increased RhoA-stimulated actomyosin contractility, and inhibition of adhesion complex turnover; however, the microtubule-dependent changes in adhesion complex composition have not been studied in a global, unbiased manner. Here we used label-free quantitative mass spectrometry-based proteomics to determine adhesion complex changes that occur upon microtubule disruption with nocodazole. Nocodazole-treated cells displayed an increased abundance of the majority of known adhesion complex components, but no change in the levels of the fibronectin-binding α5ÎČ1 integrin. Immunofluorescence analyses confirmed these findings, but revealed a change in localisation of adhesion complex components. Specifically, in untreated cells, α5-integrin co-localised with vinculin at peripherally located focal adhesions and with tensin at centrally located fibrillar adhesions. In nocodazole-treated cells, however, α5-integrin was found in both peripherally located and centrally located adhesion complexes that contained both vinculin and tensin, suggesting a switch in the maturation state of adhesion complexes to favour focal adhesions. Moreover, the switch to focal adhesions was confirmed to be force-dependent as inhibition of cell contractility with the Rho-associated protein kinase inhibitor, Y-27632, prevented the nocodazole-induced conversion. These results highlight a complex interplay between the microtubule cytoskeleton, adhesion complex maturation state and intracellular contractile force, and provide a resource for future adhesion signaling studies. The proteomics data have been deposited in the ProteomeXchange with identifier PXD001183
Cell adaptive response to extracellular matrix density is controlled by ICAP-1-dependent beta1-integrin affinity.
International audienceCell migration is an integrated process requiring the continuous coordinated assembly and disassembly of adhesion structures. How cells orchestrate adhesion turnover is only partially understood. We provide evidence for a novel mechanistic insight into focal adhesion (FA) dynamics by demonstrating that integrin cytoplasmic domain-associated protein 1 (ICAP-1) slows down FA assembly. Live cell imaging, which was performed in both Icap-1-deficient mouse embryonic fibroblasts and cells expressing active beta(1) integrin, shows that the integrin high affinity state favored by talin is antagonistically controlled by ICAP-1. This affinity switch results in modulation in the speed of FA assembly and, consequently, of cell spreading and migration. Unexpectedly, the ICAP-1-dependent decrease in integrin affinity allows cell sensing of matrix surface density, suggesting that integrin conformational changes are important in mechanotransduction. Our results clarify the function of ICAP-1 in cell adhesion and highlight the central role it plays in the cell's integrated response to the extracellular microenvironment
Validation of changes in AC components observed by MS.
<p>A) Isolated ACs were probed by Western blotting for α<sub>v</sub>-integrin, tensin-1, filamin A, ELKS and PDLIM5. B) To investigate the pattern of changes to integrins upon loss of microtubules further, HFFs treated with DMSO or nocodazole (Noc) were fixed and immunostained for α<sub>5</sub>-integrin, α<sub>v</sub>ÎČ<sub>3</sub>-integrin and vinculin and quantified as a percentage of the total cell area (line, median; box, interquartile range; whiskers, maximum and minimum; ****<i>p</i><0.0001, Student's <i>t</i>-test; <i>n</i>â=â54). C) Immunostained cells showing the cellular distribution of α<sub>5</sub>-integrin (red), α<sub>v</sub>ÎČ<sub>3</sub>-integrin (green) and vinculin (blue). Blue and yellow insets are 4Ă enlargements of corresponding boxes.</p
Protein-protein interaction network analysis of FN-enriched ventral plasma membrane complexes.
<p>Node colour was determined by a blue-red colour gradient corresponding to the log<sub>2</sub>(Noc/DMSO) values, with red nodes indicating an increase and blue nodes a decrease in protein abundance upon nocodazole treatment. Nodes were sorted according to their cell localisation and node size was proportional to the normalised spectral counts. The network was ordered according to the interaction network distance relative to the plasma membrane integrins, 1-hop, 2-hop, etc. Corresponding gene names are displayed underneath each node for clarity. FN, fibronectin; Noc, nocodazole.</p
Specificities of ÎČ1 integrin signaling in the control of cell adhesion and adhesive strength.
International audienceCells exert actomyosin contractility and cytoskeleton-dependent force in response to matrix stiffness cues. Cells dynamically adapt to force by modifying their behavior and remodeling their microenvironment. This adaptation is favored by integrin activation switch and their ability to modulate their clustering and the assembly of an intracellular hub in response to force. Indeed integrins are mechanoreceptors and mediate mechanotransduction by transferring forces to specific adhesion proteins into focal adhesions which are sensitive to tension and activate intracellular signals. α(5)ÎČ(1) integrin is considered of major importance for the formation of an elaborate meshwork of fibronectin fibrils and for the extracellular matrix deposition and remodeling. Here we summarize recent progress in the study of mechanisms regulating the activation cycle of ÎČ(1) integrin and the specificity of α(5)ÎČ(1) integrin in mechanotransduction
Changes in isolated ventral plasma membrane complexes upon microtubule disruption.
<p>A) Serum-starved HFFs plated on either fibronectin (FN) or poly-D-lysine (PDL) were treated with DMSO or nocodazole (Noc) for 4 hours, and ventral plasma membrane complexes were isolated for Western blotting for AC components (α<sub>5</sub>-integrin, talin, vinculin, paxillin, FAK, pY397-FAK and ILK) and non-AC components (BAK and transferrin receptor). B) Protein bands were quantified and normalised to FN, DMSO (mean ± SD; <i>n</i>â=â3).</p