Talin is a multifunctional cytoskeletal protein that plays a critical role in linking the actin cytoskeleton to the integrin family of transmembrane cell adhesion receptors. Two distinct integrin binding sites have been identified in talin, one present in the globular head domain (IBS1) and involved in integrin activation, and a second (IBS2), that has been delineated to a 130 residue fragment of the talin rod domain, but whose functional role is still elusive (Tremuth et al.,2004). The objective of the present study was to define the minimal structure of talin IBS2 and to investigate its functional role in the integrin-cytoskeleton connection. In the first part of this study, we used a combination of three different experimental approaches to define the minimal structure of talin IBS2: 1) an in silico bioinformatics approach to analyse sequence conservation of talin IBS2, 2) an in vivo cell biology approach to study the subcellular localization of recombinant talin fragments covering IBS2 in CHOáIIbâ3 cells, and 3) an in vitro biochemical approach consisting in protein overlay, pull down and Surface Plasmon Resonance (SPR) assays, to study the direct interaction between talin IBS2 and the integrin â3 subunit. We delineated IBS2 to a single amphipathic á-helical repeat of 23 residues within the talin rod domain. We further provided evidence that a two amino acid mutation(L2094I2095/AA) was sufficient to inactivate the IBS2 site, due to a disruption of the á helix structure, as demonstrated by infrared spectroscopy. In addition, we identified 2 lysine residues (K2085, K2089) exposed on the solvent face of á helix 50, which are directly involved in the talin IBS2-integrin interaction. In the second part of this study, we investigated the functional role of talin IBS2 in spreading defective talin (-/-) cells and showed that in contrast to full-length wild type talin, an IBS2 LI/AA mutant talin was unable to fully rescue the spread phenotype of these cells. These results provide the first direct evidence that IBS2 in the talin rod is essential to link integrins to the actin cytoskeleton.

Shigella, l’agent de la dysenterie bacillaire, envahit la muqueuse colique, causant une inflammation intense et destruction tissulaire. Afin de promouvoir l’infection, Shigella injecte des facteurs de virulence par l’intermédiaire d’un appareil de sécrétion de type III (T3SS) dans les cellules hôte, qui permettent la réorganisation du cytosquelette d’actine, régulent l’inflammation et l’intégrité du tissu. Parmi ces facteurs injectés par la bactérie, la protéine IpgD agit comme une phosphatidylinositol (PI) (4,5) bisphosphate phosphatase déphosphorylant le PIP2 en PI(5)P. L’hydrolyse du PI(4,5)P2 favorise la polymérisation de l’actine durant l’invasion des cellules épithéliales et régule négativement la migration des lymphocytes T. Il a également été démontré que le PI(5)P produit par IpgD active la voie de survie cellulaire dépendant de la PI(3) kinase et de la kinase AKT. Mon projet de thèse a pour but de caractériser le rôle d’IpgD dans les contrôles des réponses calciques durant l’invasion des cellules épithéliales par Shigella. Nous avons montré qu’IpgD limite le recrutement de récepteurs à l’inositol-triphosphate (InsP3) au site d’invasion. Des expériences d’imagerie calcique indiquent que durant les étapes précoces de l’invasion, IpgD favorise l’émission de réponses locales aux sites d’invasion de Shigella et limite celle de réponses globales. Des expériences de recouvrement de fluorescence après photo-blanchiment (FRAP) indiquent que les foyers d’invasion induits par le mutant ipgD montrent une diffusion moins restreinte que celle observée dans les foyers induits par la souche sauvage. Des études de modélisation supportent la notion qu’en limitant la production locale d’InsP3 et sa diffusion aux sites d’invasion, IpgD participe au confinement des réponses calciques locales en empêchant l’émission de réponses globales. Nous avons montré que lors de cinétiques d’infection prolongée, IpgD inhibe les réponses calciques globales dépendant de l’InsP3 induites par Shigella ou par des agonistes. L’inhibition de ces réponses conduit à un retard de l’activation de la calpaine, de la dégradation de la taline, une protéine des adhérences focales, ainsi que de la mort des cellules durant la réplication bactérienne intracellulaire. Nos résultats indiquent qu’IpgD est un effecteur critique de Shigella permettant de réguler la transition des réponses calciques locales vers des réponses globales et préservant les cellules infectées de la mort liée à une perte d’adhérence. / Shigella, the agent of bacillary dysentery, invades colonic epithelial cells where it disseminates causing an intense inflammation and tissue destruction. To efficiently promote infection, Shigella injects virulence effectors via a type III secretion system (T3SS) into host cell to divert processes involved in cytoskeletal rearrangements and processes regulating inflammatory signals or tissue integrity. Among the Shigella T3SS effectors, IpgD acts as a phosphatidylinositol (PI) (4, 5) bisphosphate phosphatase, which dephosphorylates PI (4,5) P2 to generate PI(5)P. IpgD-mediated hydrolysis of PI (4, 5) P2 favors actin polymerization during cell invasion and negatively regulates the migration of T cells. PI(5)P produced by IpgD was also shown to up-regulate the PI3K/AKT cell survival pathway and recycling of the Epidermal Growth Factor receptor. My project thesis focuses on the role of IpgD in the control of calcium responses during Shigella infection. We show that that IpgD is responsible for a decrease in the recruitment of inositol-triphosphate (InsP3) receptors at invasion sites. Ca2+-imaging experiments indicate that during the early stages of bacterial invasion, IpgD favors the elicitation of local Ca2+responses at Shigella invasion sites, and limits the induction of global Ca2+ responses. Fliuorescence Recovery After Photobleaching (FRAP) experiments indicate that actin foci induced by the ipgD mutant show faster diffusion kinetics than those in foci induced by WT. Modeling studies support the notion that the local decrease in InsP3 levels and of its diffusion kinetics triggered by IpgD at entry sites accounted for the modulation of local to global Ca2+ responses during Shigella invasion. We show that over prolonged infection kinetics, IpgD inhibits InsP3-dependent global Ca2+ responses induced by Shigella or agonists. IpgD-mediated inhibition of Ca2+ signals delays the activation of calpain, the degradation of the focal adhesion protein talin, and cell death during bacterial intracellular replication. Our results provide evidence that IpgD is a critical T3SS effector of Shigella regulating the transition from local to global Ca2+ signals, and preserving cells from death linked to loss of adhesion.

Talin is a multifunctional cytoskeletal protein that plays a critical role in linking the actin cytoskeleton to the integrin family of transmembrane cell adhesion receptors. Two distinct integrin binding sites have been identified in talin, one present in the globular head domain (IBS1) and involved in integrin activation, and a second (IBS2), that has been delineated to a 130 residue fragment of the talin rod domain, but whose functional role is still elusive (Tremuth et al.2004). The objective of the present study was to define the minimal structure of talin IBS2 and to investigate its functional role in the integrin-cytoskeleton connection.<p>In the first part of this study, we used a combination of three different experimental approaches to define the minimal structure of talin IBS2: 1) an in silico bioinformatics approach to analyse sequence conservation of talin IBS2, 2) an in vivo cell biology approach to study the subcellular localization of recombinant talin fragments covering IBS2 in CHOáIIbâ3 cells, and 3) an in vitro biochemical approach consisting in protein overlay, pull down and Surface Plasmon Resonance (SPR) assays, to study the direct interaction between talin IBS2 and the integrin â3 subunit. We delineated IBS2 to a single amphipathic á-helical repeat of 23 residues within the talin rod domain. We further provided evidence that a two amino acid mutation(L2094I2095/AA) was sufficient to inactivate the IBS2 site, due to a disruption of the á helix structure, as demonstrated by infrared spectroscopy. In addition, we identified 2 lysine residues (K2085, K2089) exposed on the solvent face of á helix 50, which are directly involved in the talin IBS2-integrin interaction.<p>In the second part of this study, we investigated the functional role of talin IBS2 in spreading defective talin (-/-) cells and showed that in contrast to full-length wild type talin, an IBS2 LI/AA mutant talin was unable to fully rescue the spread phenotype of these cells. These results provide the first direct evidence that IBS2 in the talin rod is essential to link integrins to the actin cytoskeleton. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

The mechanical properties of the tissue vary widely around the body, from the soft brain to the rigid bone. Tissue cells are able to sense mechanical signals from their environment, which influence many aspects of cell behaviour such as migration, proliferation and differentiation. Focal adhesions (FAs) are large protein complexes that form the bridge between the extracellular matrix (ECM)-binding integrins and the contractile actin cytoskeleton. Here, they sense the rigidity of the local environment and translate this information into a cellular response, a process known as mechanotransduction. However, the FA proteins required for mechanotransduction, and the molecular mechanisms involved in this fundamental process, remain to be elucidated. Talin, vinculin, FAK and paxillin are four core FA-associated proteins that are thought to be involved in mechanotransduction. These proteins associate and dissociate from the complex in a constant state of flux. Using a live-cell imaging approach, I found that the rate of dynamic exchange of an FA protein correlates to its function. The FA appears to have a modular organisation; the slowest proteins have a structural role, such as talin and vinculin, responsible for directly linking integrin to actin and sensing the ECM stiffness. The signalling proteins are turned over more rapidly, including FAK and paxillin, and are responsible for directing the cellular response to force-generated signals from the ECM.The second results chapter focused on the force-dependent interactions between talin, vinculin and actin. The talin domains R2R3 were identified as the key mechanosensitive vinculin-binding sites, which are exposed upon the application of force across the talin rod. Vinculin binding to R2R3 led to actin associating with the central actin-binding site in the talin rod (ABS2), which is required for the transmission of actomyosin tension onto the underlying substrate as cellular traction force. Finally, the protein turnover data were incorporated into two mathematical models, describing talin and vinculin turnover, which were able to simulate the dynamic exchange of various talin and vinculin mutants in response to changing ECM stiffness. Using these models, the talin ABS2-actin and vinculin tail-actin interactions were found to be extremely important for sensing the stiffness of the ECM. These findings significantly increase our knowledge of the molecular mechanisms underpinning cellular mechanotransduction. Increased understanding of how mechanical signals are sensed and interpreted by the cell could lead to a number of novel therapies for a wide range of associated diseases, such as atherosclerosis, muscular dystrophy and cancer.

No description available.

The survival of breast cancer patients declines when tumors are invasive and have an increased possibility of metastasizing to distal sites. Transforming Growth Factor-beta (TGF-beta) suppresses breast cancer formation by preventing cell cycle progression in mammary epithelial cells. However, at late stage of mammary carcinogenesis, due to genetic and epigenetic alterations, TGF-beta loses its cytostatic actions, and contributes to tumor invasion by promoting cell proliferation, Actin cytoskeletal reorganization, as well as Epithelial to Mesenchymal Transition (EMT). Despite the key role of TGF-beta1 in tumor suppression as well as tumor progression, the molecular mechanisms underlying the conversion of TGF-beta form an inhibitor of proliferation in mammary breast cancer cells to an inducer of their cell growth and EMT have not been fully elucidated. Thus, acquiring a basic knowledge on the mechanism of TGF-beta regulating its target genes and its contribution to cancer progression may highlight new avenues for cancer therapy development. This prompted us to further investigate and identify TGF-beta-inducible genes that may be involved in TGF-beta biological responses during tumorigenesis. / In this thesis, we identified Talin as a novel TGF-beta1 target gene that acts as an antagonist to inhibit TGF-beta-mediated cell growth arrest and transcriptional activity in mammary cancer cell line, MCF-7. Searching for new partners of activated Smads, we found that TGF-beta1 induces Talin translocation from cytosol to the plasma membrane where Talin physically interacts with the TGF-beta1 signaling components, the Smads and the receptors. Furthermore, we observed that TGF-beta1 stimulation leads to the formation of Actin stress fibers where Talin was detected at the end of these stress fibers. Taken all together, the obtained data show that TGF-beta1 positively induced expression of Talin and suggests a role for Talin, which acts as a negative feedback loop to control TGF-beta biological responses.

Formation of a tubular organ, such as the heart, requires cells to integrate positional and polarity signals in order to enclose a fluid or gas transporting lumen. Developing tubes must establish a site for lumen initiation, demarcate membrane domains, and modulate cell polarization and morphology. The Drosophila melanogaster embryonic heart is a mesodermal tube model displaying a unique polarity, reminiscent of vertebrate vasculature. We have characterized a role for the transmembrane adhesion receptor αPS3βPS1 integrin and its cytoplasmic adaptor Talin in heart tubulogenesis. βPS1 and Talin are early indicators of the luminal site and Talin-mediated integrin function is essential for cardioblast polarization and morphology prior to and during lumen development. Careful analysis of hearts in embryos homozygous for a null allele of rhea, the gene encoding Talin, reveals that Talin is required to correctly orient the heart cell polarity such that a continuous central open lumen is enclosed. Without proper integrin or Talin function, the luminal determinants Slit and its receptor Robo are not stabilized within the heart, a central lumen fails to form, and the midline is instead marked by continuous adhesion. Furthermore, although Talin is essential for proper βPS1 integrin localization within the heart, either of Talin’s two integrin binding sites are sufficient to stabilize βPS1 along the luminal domain and establish an open cardiac tube. Taken together, our findings reveal an instructive role for integrins and Talin in communicating polarization cues central to heart tubulogenesis. / Thesis / Doctor of Philosophy (PhD)

No description available.

No description available.

Integrins are large heterodimeric (αβ) cell surface receptors that play a key role in the formation of focal adhesion complexes and are involved in various signal transduction pathways. They are ‘activated’ to a high affinity state by the formation of an intracellular complex between the membrane, the integrin β-subunit tail and talin, a process known as ‘inside-out activation’. The head domain of talin, a FERM domain homologue, plays a vital role in the formation of this complex. Recent studies also suggest that kindlins act in synergy with talin to induce integrin activation. Despite much available structural and functional data, details of how talin activates integrins remain elusive. In this thesis a multiscale simulation approach (using a combination of coarse-grained and atomistic molecular dynamics simulations) together with NMR experiments were employed to study talin-mediated integrin inside-out activation. A number of novel insights emerged from these studies including: (i) the crucial role of negatively charged lipids in talin/membrane association; (ii) a novel V-shape conformation of the talin head domain which optimizes its interactions with negatively charged lipids; (iii) that interactions of talin with negatively charged moieties in the membrane orient talin to optimize interactions with the β cytoplasmic tail; (iv) that binding of talin to the β cytoplasmic tail promotes rearrangement of the integrin TM helices and weakens the integrin α/β association; and (v) that an increase in the tilt angle of the β integrin TM helix initiates a scissoring movement of the two integrin TM helices. These results, combined with experimental data, provide new insights into the mechanism of integrin inside-out activation. The same multiscale approach was used to demonstrate the crucial role of the Gx3G motif in the packing of the integrin transmembrane helices. Using recent structural data the integrin/talin complex was modelled and inserted in bilayers which resemble the biological plasma membrane. The results demonstrate the dynamic nature of the integrin receptor and suggest that the integrin/talin complex alters the lipid organization and motion in the outer and inner bilayer leaflets in an asymmetric way and that diffusion of lipids in the vicinity of the protein is slowed down. The work in this thesis demonstrates that multiscale simulations have considerable strengths when applied to investigations of structure/function relationships in membrane proteins.