Myosin-X provides a motor-based link between integrins and the cytoskeleton (original) (raw)
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Myosin-X and talin modulate integrin activity at filopodia tips
2020
Filopodia assemble unique integrin-adhesion complexes to sense the extracellular matrix. However, the mechanisms of integrin localization and regulation in filopodia are poorly defined. Here, we observed that active integrins accumulate at the tip of myosin-X(MYO10)-positive filopodia while inactive integrins are uniformly distributed. RNAi depletion of 10 integrin activity modulators identified talin as the principal integrin activator in filopodia. Deletion of the MYO10-FERM domain, or mutation of the β1-integrin-binding residues within, revealed MYO10 as facilitating integrin activation but not transport in filopodia. However, MYO10-FERM alone could not activate integrins, potentially due to dual binding to both a- and β-integrin tails. As swapping MYO10-FERM with talin-FERM enabled integrin activation in filopodia, our data indicate that an integrin-binding FERM domain coupled to a myosin motor is a core requirement for integrin activation in filopodia. Therefore, we propose a t...
Nature cell biology, 2013
How different integrins that bind to the same type of extracellular matrix protein mediate specific functions is unclear. We report the functional analysis of β 1 -and α v -class integrins expressed in pan-integrin-null fibroblasts seeded on fibronectin. Reconstitution with β 1 -class integrins promotes myosin-II-independent formation of small peripheral adhesions and cell protrusions, whereas expression of α v -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 α v -class integrins accumulating in adhesion areas exposed to high traction forces. Quantitative proteomics linked α v -class integrins to a GEF-H1-RhoA pathway coupled to the formin mDia1 but not myosin II, and α 5 β 1 integrins to a RhoA-Rock-myosin II pathway. Our study assigns specific functions to distinct fibronectin-binding integrins, demonstrating that α 5 β 1 integrins accomplish force generation, whereas α v -class integrins mediate the structural adaptations to forces, which cooperatively enable cells to sense the rigidity of fibronectin-based microenvironments.
Journal of Biological Chemistry, 1998
The ␣ 5  1 integrin is a cell surface receptor for fibronectin implicated in several cellular activities including cell proliferation, differentiation, and migration. The primary site at which the ␣ 5  1 integrin interacts with fibronectin is the RGD (Arg-Gly-Asp) amino acid sequence. In general, the sites on the integrin ␣ subunits involved in ligand binding are not well characterized. Based on previous cross-linking studies, sequence alignment, predicted conformation, and intron-exon boundaries, we identified a 144-residue region (positions 223-367) on the ␣ 5 subunit as a putative binding region and divided it into four subdomains named domains I, II, III, and IV. Chimeric receptors were prepared in which sequences on the ␣ 5 subunit were exchanged with the corresponding sequences on the ␣ 6 subunit, which is specific for laminin and does not bind via an RGD sequence. The mutated human ␣ 5 integrin gene was transfected into CHO B2 cells, which are deficient in ␣ 5 expression. Only chimeras of domain III or IV express on the cell surface. Both of these chimeras decreased the adhesion, spreading, focal adhesion assembly, and migration on fibronectin. The adhesion of the chimeric receptors to fibronectin remained sensitive to the RGD peptide, and antibodies that inhibit interaction with the fibronectin synergy site and RGD loop remain inhibitory for the chimeras, indicating that our chimeras do not inhibit binding to either the RGD or synergy sites. Finally, the affinity of soluble fibronectin to cells via the ␣ 5  1 receptor decreased only about 3-fold. This decrease is substantially less than the observed effects on migration and spreading, which were not altered by changes in substrate concentration. Thus, the alteration in binding sites does not easily account for the changes in cell spreading and focal adhesion assembly. The tyrosine phosphorylation and focal adhesion assembly that are seen when cells expressing the wild type ␣ 5 receptor adhere to fibronectin were inhibited in cells expressing the chimeric receptors. Therefore, our results suggest that the chimeras of these domains likely interrupt ␣ 5 -mediated conformational signaling. The abbreviations used are: mAb, monoclonal antibody; CHO, Chinese hamster ovary; CMF-HH buffer, Ca 2ϩ -, Mg 2ϩ -free Hepes-Hanks' buffer; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; BSA, bovine serum albumin; ICAM, intercellular adhesion molecule; VCAM, vascular cell adhesion molecule.
Integrin alpha3 subunit participates in myoblast adhesion and fusion in vitro
Differentiation, 2006
Satellite cells are myogenic precursor cells, participating in growth, and regeneration of skeletal muscles. The proteins that play a role in myogenesis are integrins. In this report, we show that the integrin a3 subunit is expressed in quiescent satellite cells and activated myoblasts. We also find that in myoblasts the integrin a3 subunit is localized at cell-cell and cell-extracellular matrix contacts. We notice that increase in protein and mRNA encoding the integrin a3 subunit accompanies myoblast differentiation. Using double immunofluorescence and immunoprecipitation experiments, we demonstrate that the integrin a3 subunit colocalizes with actin, and binds the integrin b1 subunit and ADAM12, suggesting that the complex a3b1/ ADAM12 is probably involved in myoblast fusion. Importantly, overexpression of the full-length integrin a3 subunit increases myoblast fusion whereas an antibody against its extracellular domain inhibits fusion. These data demonstrate that the integrin a3 subunit may contribute to satellite cell activation and then myoblast adhesion and fusion.
Characterization of β1 Integrin Cytoplasmic Domain Binding Proteins
2008
The extracellular matrix (ECM) provides the structural frame for the development of tissues and organs. The ECM is bound by numerous membranous matrix-adhesion molecules and thereby triggers intracellular signals that control various cellular functions such as survival, polarity, proliferation and differentiation. Integrins represent an important family of ECM adhesion molecules which link the ECM with the intracellular actin-cytoskeleton. Integrin mediated adhesion structures also serve as important signaling platforms, although the integrin itself does not harbors any catalytic domains. Therefore integrin signaling depends on the recruitment of a number of cytoplasmic proteins that directly or indirectly bind to the short cytoplasmic integrin tails. During my PhD thesis I worked on three of these molecules, ILK, Kindlins and Palladin, and used the mouse as a model system to address their in vivo function. First, I investigated the role of integrin-linked kinase (ILK) in skeletal m...
Integrin adhesion receptors are structurally dynamic proteins that adopt a number of functionally relevant conformations. We have produced a conformation-dependent anti-α 5 monoclonal antibody (SNAKA51) that converts α 5 β 1 into a ligand-competent form and promotes fibronectin binding. In adherent fibroblasts, SNAKA51 preferentially bound to integrins in fibrillar adhesions. Clustering of integrins expressing this activation epitope induced directional translocation of α 5 β 1 , mimicking fibrillar adhesion formation. Priming of α 5 β 1 by SNAKA51 increased the accumulation of detergent-resistant fibronectin in the extracellular matrix, thus identifying an integrin conformation that promotes matrix assembly. The SNAKA51 epitope was mapped to the calf-1/calf-2 domains. We propose that the action of the antibody causes the legs of the integrin to change conformation and thereby primes the integrin to bind ligand. These findings identify SNAKA51 as the first anti-integrin antibody to selectively recognize a subset of adhesion contacts, and they identify an integrin conformation associated with integrin translocation and fibronectin matrix formation.
Functional Role of the Cytoplasmic Domain of the Integrin or5 Subunit
1993
The purpose of this study was to explore the functional role of the cytoplasmic domain of the ct subunit of the o~5//31 integrin, a fibronectin receptor. Mutant CHO cells that express very low levels of endogenous hamster a5 subunit (CHO clone B2) were transfected with an expression vector containing fulllength or truncated human a5 cDNAs to form chimeric human o~5/hamster/~1 integrins. Three transfectants were examined: B2a27 expresses a full-length human c~5 subunit with 27 amino acids in the cytoplasmic domain; B2al0 expresses an or5 with a 17amino acid cytoplasmic truncation; B2al expresses an or5 with a 26-amino acid truncation. Levels of ot5//~l surface expression in B2a27 and B2al0 cells were similar to that in wild type CHO cells. The expression of ct5//31 in B2al cells was less, amounting to 15-20% of WT levels, despite message levels that were three to five times greater than those of B2a27. The transfectants were used to examine the role of the or5 cytoplasmic domain in cell adhesion, cell motility, cytoskeletal organization, and integrin-mediated tyrosine phosphorylation.