The Talin Head Domain Reinforces Integrin-Mediated Adhesion by Promoting Adhesion Complex Stability and Clustering

Talin serves an essential function during integrin-mediated adhesion in linking integrins to actin via the intracellular adhesion complex. In addition, the N-terminal head domain of talin regulates the affinity of integrins for their ECM-ligands, a process known as inside-out activation. We previously showed that in Drosophila, mutating the integrin binding site in the talin head domain resulted in weakened adhesion to the ECM. Intriguingly, subsequent studies showed that canonical inside-out activation of integrin might not take place in flies. Consistent with this, a mutation in talin that specifically blocks its ability to activate mammalian integrins does not significantly impinge on talin function during fly development. Here, we describe results suggesting that the talin head domain reinforces and stabilizes the integrin adhesion complex by promoting integrin clustering distinct from its ability to support inside-out activation. Specifically, we show that an allele of talin containing a mutation that disrupts intramolecular interactions within the talin head attenuates the assembly and reinforcement of the integrin adhesion complex. Importantly, we provide evidence that this mutation blocks integrin clustering in vivo. We propose that the talin head domain is essential for regulating integrin avidity in Drosophila and that this is crucial for integrin-mediated adhesion during animal development

A conserved lipid-binding loop in the kindlin FERM F1 domain is required for kindlin-mediated aIIbB3 integrin coactivation

The activation of heterodimeric integrin adhesion receptors from low to high affinity states occurs in response to intracellular signals that act on the short cytoplasmic tails of integrin beta subunits. Binding of the talin FERM (four-point-one, ezrin, radixin, moesin) domain to the integrin beta-tail provides one key activation signal, but recent data indicate that the kindlin family of FERM domain proteins also play a central role. Kindlins directly bind integrin beta subunit cytoplasmic domains at a site distinct from the talin-binding site, and target to focal adhesions in adherent cells. However, the mechanisms by which kindlins impact integrin activation remain largely unknown. A notable feature of kindlins is their similarity to the integrin-binding and activating talin FERM domain. Drawing on this similarity, here we report the identification of an unstructured insert in the kindlin F1 FERM domain, and provide evidence that a highly conserved polylysine motif in this loop supports binding to negatively charged phospholipid head groups. We further show that the F1 loop and its membrane-binding motif are required for kindlin-1 targeting to focal adhesions, and for the cooperation between kindlin-1 and -2 and the talin head in aIIbB3 integrin activation, but not for kindlin binding to integrin beta tails. These studies highlight the structural and functional similarities between kindlins and the talin head and indicate that as for talin, FERM domain interactions with acidic membrane phospholipids as well beta-integrin tails contribute to the ability of kindlins to activate integrins

Structure of a double ubiquitin-like domain in the talin head: a role in integrin activation

Talin is a 270-kDa protein that activates integrins and couples them to cytoskeletal actin. Talin contains an N-terminal FERM domain comprised of F1, F2 and F3 domains, but it is atypical in that F1 contains a large insert and is preceded by an extra domain F0. Although F3 contains the binding site for β-integrin tails, F0 and F1 are also required for activation of β1-integrins. Here, we report the solution structures of F0, F1 and of the F0F1 double domain. Both F0 and F1 have ubiquitin-like folds joined in a novel fixed orientation by an extensive charged interface. The F1 insert forms a loop with helical propensity, and basic residues predicted to reside on one surface of the helix are required for binding to acidic phospholipids and for talin-mediated activation of β1-integrins. This and the fact that basic residues on F2 and F3 are also essential for integrin activation suggest that extensive interactions between the talin FERM domain and acidic membrane phospholipids are required to orientate the FERM domain such that it can activate integrins

Etude de la voie de signalisation activatrice des intégrines beta2 et beta3 dans les neutrophiles et les plaquettes

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

In Silico Study for Algerian Essential Oils as Antimicrobial Agents against Multidrug-Resistant Bacteria Isolated from Pus Samples

In the context of the globally growing problem of resistance to most used antibacterial agents, essential oils offer promising solutions against multidrug-resistant (MDR) bacterial pathogens. The present study aimed to evaluate the prevalence, etiology, and antibiotic-resistance profiles of bacteria responsible for pyogenic infections in Regional Military University Hospital of Constantine. Disc diffusion and broth microdilution (MIC) methods were used to evaluate the antimicrobial activity of essential oils from five Algerian aromatic plants growing wild in the north of Algeria—Salvia officinalis (Sage), Thymus vulgaris (Thyme), Mentha pulegium L. (Mentha), Rosmarinus officinalis (Rosemary), and Pelargonium roseum (Geranium)—against reference and MDR strains. During three months of the prospective study, 112 isolates out of 431 pus samples were identified. Staphylococcus aureus was the most predominant species (25%), followed by Klebsiella pneumoniae (21.42%), Pseudomonas aeruginosa (21%), and Escherichia coli (17.95%). Among pus isolates, 65 were MDR (58.03%). The radial streak-line assay showed that R. officinalis and M. pulegium L. had weak activity against the tested strains, whereas P. roseum showed no activity at all. Meanwhile, T. vulgaris was the most potent, with an inhibition zone of 12–26 mm and an MIC value ranging between 0.25 and 1.25%, followed by S. officinalis with an inhibition zone of 8–12 mm and an MIC value ranging between 0.62 and 2.5%. Generally, A. baumannii and S. aureus ATCC6538P were the most sensitive strains, whereas P. aeruginosa ATCC27853 was the most resistant strain to the oils. Gas chromatography–mass spectrometry analysis of chemical composition revealed the presence of borneol (76.42%) and thymol (17.69%) as predominant in thyme, whereas camphor (36.92%) and α- thujone (34.91%) were the major volatiles in sage. The in-silico study revealed that sesquiterpenes and thymol had the highest binding free energies against the vital enzymes involved in biosynthesis and repair of cell walls, proteins, and nucleic acids compared to monoterpenes. The results demonstrated that T. vulgaris and S. officinalis are ideal candidates for developing future potentially active remedies against MDR strains

The E3 ubiquitin ligase specificity subunit ASB2 targets filamins for proteasomal degradation by interacting with the filamin actin-binding domain

International audienceFilamins are an important family of actin-binding and crosslinking proteins that mediate remodeling of the actin cytoskeleton and maintain extracellular matrix connections by anchoring transmembrane proteins to actin filaments and linking them to intracellular signaling cascades. We recently found that filamins are targeted for proteasomal degradation by the E3 ubiquitin ligase specificity subunit ASBα and that acute degradation of filamins through this ubiquitin-proteasome pathway correlates with cell differentiation. Specifically, in myeloid leukemia cells retinoic-acid-induced expression of ASB2α triggers filamin degradation and recapitulates early events crucial for cell differentiation. ASB2α is thought to link substrates to the ubiquitin transferase machinery; however, the mechanism by which ASB2α interacts with filamin to induce degradation remained unknown. Here, we use cell-based and biochemical assays to show that the subcellular localization of ASB2α to actin-rich structures is dependent on filamin and that the actin-binding domain (ABD) of filamin mediates the interaction with ASB2α. Furthermore, we show that the ABD is necessary and sufficient for ASB2α-mediated filamin degradation. We propose that ASB2α exerts its effect by binding the ABD and mediating its polyubiquitylation, so targeting filamins for degradation. These studies provide the molecular basis for ASB2α-mediated filamin degradation and unravel an important mechanism by which filamin levels can be acutely regulated

Integrin-binding to the talin head, but not integrin activation, is required for muscle attachment.

<p>(<b>a</b>) Schematic of key domains in talin for this study. The talin head is contains an N-terminal atypical FERM domain <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004756#pgen.1004756-Elliott1" target="_blank">[10]</a> and a C-terminal rod domain comprised of 13 helical bundles <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004756#pgen.1004756-Goult1" target="_blank">[59]</a>. (<b>b</b>) Alignment of residues 325–375 of fly talin F3 domain with human talin isoforms. Dark blue indicates identical residues between homologues, lighter blue indicates similar residues. The mutations utilized to study integrin activation are indicated with an arrowhead. (<b>c–e</b>) Integrin-dependent phenotypes germband retraction (c), dorsal closure (d) and muscle attachment (e) were assayed in talin-null embryos, WT talinGFP-rescued embryos, and talinGFP*L334R-rescued embryos. Apart from mild muscle detachment in about 20% of embryos, the talinGFP*L334R transgene was able to rescue all phenotypes such that the embryos hatched to the larval stages. (<b>f–g</b>) Maternal zygotic talin null embryos rescued with either full-length WT talinGFP transgene (f) or talinGFP*L334R mutant transgene (g) and stained for F-actin (green) and βPS-integrin (magenta). (<b>h–j</b>) MTJs of talin null embryos rescued with either talinGFP-WT (<b>h</b>), talinGFP*R367A (<b>i</b>), talinGFP*L334R (<b>j</b>). Embryos were stained for anti- <b>α</b>PS2-integrin (green in <b>h–j</b>; grey in <b>h′–j′</b>) and tiggrin, a Drosophila ECM molecule (red in <b>h–j</b>). (<b>h″–j″</b>) Average intensity profiles for integrin and tiggrin across the widths of the boxed areas in h–j. Tiggrin and integrin completely overlapped at MTJs in WT talin rescue embryos (h″), but were separated from one another in talin-null embryos rescued with talinGFP*R367A (i–i″). Overlap between tiggrin and integrin was maintained in talin-null embryos rescued with talinGFP*L334R (j–j″). The pink arrowheads mark the sites of separated integrin and ECM signal. (<b>k</b>) Activation of human integrins by fly talin head constructs was measured in CHO cells. The L334R mutation was sufficient to abrogate integrin activation. (<b>l–m</b>) Recruitment of ubi-promoter driven full-length WT talinGFP and talinGFP*L334R to sites of adhesion was assayed in talin null (l) and in wild-type embryos (m). Compared to WT TalinGFP, TalinGFP*L334R was well recruited in a background devoid of any endogenous talin (l–l″; **p<0.01), but competed less well in the presence of endogenous talin and was only weakly recruited to sites of adhesion compared to WT, which was robustly recruited (m–m″; ***p<0.001). (<b>n</b>) FRAP experiments on WT talinGFP and talinGFP*L334R reveal that talinGFP*L334R is much less stable at sites of adhesion than WT talinGFP. Scale bars: f–g  = 100 µm; h–j;l–m = 20 µm.</p

The talin head is essential for integrin function in <i>Drosophila</i>.

<p>(<b>a–c</b>) Maternal-zygotic talin null embryos (shown in a) rescued with either full-length WT talinGFP transgene (b) or headless talinGFP transgene (c) and stained for F-actin (green) and integrin (magenta). (<b>d–f</b>) Integrin-dependent phenotypes germband retraction (d), dorsal closure (e) and muscle attachment (f) were assayed in talin-null embryos, WT-talin-rescued embryos, and headless-talin-rescued embryos. The talin head was required for all three processess assayed. Scale bar = 100 µm. (<b>g–h</b>) Recruitment of ubi-promoter driven, GFP-tagged full-length WT talin and headless talin to sites of adhesion was assayed in wild-type embryos (g) and in a talin null background (h). In a WT background, headless-talinGFP competed less well with endogenous talin and was only weakly recruited to sites of adhesion compared to WT (***p<0.001); in the absence of endogenous talin, headless-talin was well recruited to sites of adhesion. (<b>i</b>) FRAP experiments on talinGFP-WT and headless-talinGFP reveal that headless talin is much less stable at sites of adhesion than talinGFP-WT. (<b>j–m</b>) Confocal z-stacks of stage 17 maternal/zygotic-mutant embryos rescued with either full length WT talin (j,l) or headless-talin (k,m). (<b>j–k</b>) adhesions stained for talin (green in j–k; grey in j′–k′) and integrin (red in j–k). In the absence of the talin head, talin was still well recruited. (<b>l–m</b>) Muscles stained for talin (magenta in l–m) and paxillin (green in l–m; grey in l′–m′). Paxillin was not well recruited to adhesions. Scale bars: a–e = 100 µm; j–m = 20 µm.</p