Rôle du phosphatidylinositol 5-phosphate dans la survie et la migration cellulaires : implication dans la dynamique des membranes et du cytosquelette

Les phosphoinositides jouent un rôle essentiel dans le contrôle spatio-temporel de grandes voies de signalisation impliquées dans la prolifération, la survie, la dynamique du cytosquelette, le trafic ou la migration. Le phosphatidylinositol 5-phosphate (PtdIns5P) est le dernier membre de la famille à avoir été caractérisé et ses fonctions ne sont pas encore totalement connues. Dans ce travail, nous avons étudié le rôle de ce second messager lipidique dans la signalisation en relation avec la survie (régulation de la voie PI3-kinase/Akt) et la migration (activation des GTPases de la famille Rho). Dans un premier modèle, nous avons étudié les processus invasifs des lymphomes anaplasiques à grandes cellules exprimant la protéine chimérique NPM-ALK au fort pouvoir transformant. Nous avons montré que la dissémination de ces lymphomes dépendait de la GTPase Rac1 qui orchestre la structuration d'invadopodes fonctionnels en régulant la formation du complexe CD44/MMP-9/Hsp90 sur la surface des cellules NPM-ALK(+). CD44 ancre le complexe à la surface des cellules et Hsp90 est requise pour l'activation extracellulaire de MMP-9. De façon intéressante, nous avons mesuré des taux élevés de PtdIns5P dans les cellules NPM-ALK(+), qui dans ce modèle est produit par la lipide kinase PIKfyve activée par NPM-ALK. L'inhibition de PIKfyve conduit à un blocage de l'invasion associé à une modification de la répartition de MMP-9 à la surface cellulaire, ainsi qu'à un défaut de sa maturation, suggérant que le PtdIns5P serait central à l'invasion des lymphomes NPM-ALK(+). En parallèle, nous avons étudié le PtdIns5P dans l'infection par la bactérie pathogène Shigella flexneri responsable de la dysenterie chez l'homme. L'équipe avait montré que le facteur de virulence IpgD transformait le PtdIns(4,5)P2 en PtdIns5P, ce qui conduisait à une activation de la voie de survie PI 3-Kinase/Akt. Nous avons montré que le PtdIns5P active le récepteur à l'EGF (EGFR)indépendamment de son ligand, et que ce dernier est requis pour l'activation d'Akt. De plus, le PtdIns5P agit en perturbant le trafic de l'EGFR qui n'est plus dégradé suite à son activation mais plutôt recyclé pour maintenir la cellule activée. Dans ce modèle, le PtdIns5P modulerait les propriétés de diffusion de la membrane plasmique pour conduire à l'activation du récepteur. Par ailleurs, nous avons montré que le PtdIns5P activait les GTPases Rac1 (en liant directement son facteur d'échange Tiam1)et Cdc42 en périphérie cellulaire où cette dernière pourrait jouer un rôle dans la dynamique des membranes requises pour l'activation sans ligand du récepteur. Le module Tiam1/Rac1 répond par ailleurs à des taux élevés de PtdIns5P en activant la dynamique de l'actine de façon polarisée et en se localisant au niveau des endosomes précoces où nous mesurons une accumulation de PtdIns5P, ce qui induit un chimiotactisme fort en réponse à un gradient de sérum. Ces données permettent de proposer un rôle du PtdIns5P dans l'intégration entre le trafic et la dynamique de l'actine pour favoriser les voies de recyclage de récepteurs pour réguler la survie et l'invasion.Phosphoinositides are key regulators of signalling pathway involved in proliferation, survival, cytoskeleton organization, vesicular trafficking and migration. Phosphatidylinositol 5-phosphate (PtdIns5P) is the last characterized and its metabolism and cellular functions are still elusive. In this work, we focused on the potential role of this lipid second messenger in signalling regulating survival (regulation of the PI 3-Kinase pathway) and migration (activation of Rho family GTPases). In a first model, we studied the invasiveness of anaplastic large cell lymphomas (ALCL) expressing the chimeric tyrosine kinase oncogene NPM-ALK, which drives malignant transformation of 75% of ALCLs. We showed that dissemination of these lymphomas is dependent on the GTPase Rac1, which promotes the structuration of functional invadopodia by regulating the formation of the CD44/MMP-9/Hsp90 complex at the surface of NPM-ALK(+) cells. CD44 anchors the complex on the cell surface, and Hsp90 is required for the extracellular activation of MMP-9. Interestingly, we measured elevated levels of PtdIns5P in NPM-ALK(+) cells and its production is dependent on the NPM-ALK-activated PtdIns 5-Kinase, PIKfyve. Inhibition of PIKfyve strongly reduces the invasive capacities by disturbing MMP-9 cell surface distribution, which leads to defects in its activation, suggesting that PtdIns5P plays a central role in the srpeading of NPM-ALK(+) lymphomas. In parallel, we studied the role of PtdIns5P during Shigella flexneri infection, which is responsible for the dysentery in humans. Our group previously showed that the virulence effector IpgD transforms the PtdIns(4,5)P2 into PtdIns5P, and that this conversion drives the activation of the PI 3-Kinase/Akt survival pathway. We found that the EGF receptor is the key intermediate between PtdIns5P and Akt activation. PtdIns5P, that we found accumulated on early endosomes, induces modifications of trafficking, which abolishes EGFR degradation and promotes its recycling towards the plasma membrane. In this model, PtdIns5P modulates the diffusion properties of the plasma membrane to promote receptor activation without ligand. In addition, we showed that PtdIns5P activates the Rac1 GTPases, by binding to the PH domain of its exchange factor Tiam1. Cdc42 also gets activated at the cell periphery where it could be involved in the membrane dynamic required for the activation of receptors without ligands and the subsequent internalization. The Tiam1/Rac1 module responds to elevated levels of PtdIns5P by inducing a strong polarized actin dynamics, leading to chimiotactism in response to serum gradients, and is an important component of anaplastic lymphomas metastatic process. This work points to a crucial role of PtdIns5P in the integration between traffic and actin dynamic to favour the recycling pathway of receptors to regulate the survival and invasion

Stabilization of non-admissible curves for a class of nonholonomic systems

The problem of tracking an arbitrary curve in the state space is considered for underactuated driftless control-affine systems. This problem is formulated as the stabilization of a time-varying family of sets associated with a neighborhood of the reference curve. An explicit control design scheme is proposed for the class of controllable systems whose degree of nonholonomy is equal to 1. It is shown that the trajectories of the closed-loop system converge exponentially to any given neighborhood of the reference curve provided that the solutions are defined in the sense of sampling. This convergence property is also illustrated numerically by several examples of nonholonomic systems of degrees 1 and 2.Comment: This is the author's version of the manuscript accepted for publication in the Proceedings of the 2019 European Control Conference (ECC'19

Transmission of integrin β7 transmembrane domain topology enables gut lymphoid tissue development.

Integrin activation regulates adhesion, extracellular matrix assembly, and cell migration, thereby playing an indispensable role in development and in many pathological processes. A proline mutation in the central integrin β3 transmembrane domain (TMD) creates a flexible kink that uncouples the topology of the inner half of the TMD from the outer half. In this study, using leukocyte integrin α4β7, which enables development of gut-associated lymphoid tissue (GALT), we examined the biological effect of such a proline mutation and report that it impairs agonist-induced talin-mediated activation of integrin α4β7, thereby inhibiting rolling lymphocyte arrest, a key step in transmigration. Furthermore, the α4β7(L721P) mutation blocks lymphocyte homing to and development of the GALT. These studies show that impairing the ability of an integrin β TMD to transmit talin-induced TMD topology inhibits agonist-induced physiological integrin activation and biological function in development

Rap1 binding and a lipid-dependent helix in talin F1 domain promote integrin activation in tandem.

Rap1 GTPases bind effectors, such as RIAM, to enable talin1 to induce integrin activation. In addition, Rap1 binds directly to the talin1 F0 domain (F0); however, this interaction makes a limited contribution to integrin activation in CHO cells or platelets. Here, we show that talin1 F1 domain (F1) contains a previously undetected Rap1-binding site of similar affinity to that in F0. A structure-guided point mutant (R118E) in F1, which blocks Rap1 binding, abolishes the capacity of Rap1 to potentiate talin1-induced integrin activation. The capacity of F1 to mediate Rap1-dependent integrin activation depends on a unique loop in F1 that has a propensity to form a helix upon binding to membrane lipids. Basic membrane-facing residues of this helix are critical, as charge-reversal mutations led to dramatic suppression of talin1-dependent activation. Thus, a novel Rap1-binding site and a transient lipid-dependent helix in F1 work in tandem to enable a direct Rap1-talin1 interaction to cause integrin activation

A RIAM/lamellipodin-talin-integrin complex forms the tip of sticky fingers that guide cell migration.

The leading edge of migrating cells contains rapidly translocating activated integrins associated with growing actin filaments that form 'sticky fingers' to sense extracellular matrix and guide cell migration. Here we utilized indirect bimolecular fluorescence complementation to visualize a molecular complex containing a Mig-10/RIAM/lamellipodin (MRL) protein (Rap1-GTP-interacting adaptor molecule (RIAM) or lamellipodin), talin and activated integrins in living cells. This complex localizes at the tips of growing actin filaments in lamellipodial and filopodial protrusions, thus corresponding to the tips of the 'sticky fingers.' Formation of the complex requires talin to form a bridge between the MRL protein and the integrins. Moreover, disruption of the MRL protein-integrin-talin (MIT) complex markedly impairs cell protrusion. These data reveal the molecular basis of the formation of 'sticky fingers' at the leading edge of migrating cells and show that an MIT complex drives these protrusions

sigReannot: an oligo-set re-annotation pipeline based on similarities with the Ensembl transcripts and Unigene clusters

<p>Abstract</p> <p>Background</p> <p>Microarray is a powerful technology enabling to monitor tens of thousands of genes in a single experiment. Most microarrays are now using oligo-sets. The design of the oligo-nucleotides is time consuming and error prone. Genome wide microarray oligo-sets are designed using as large a set of transcripts as possible in order to monitor as many genes as possible. Depending on the genome sequencing state and on the assembly state the knowledge of the existing transcripts can be very different. This knowledge evolves with the different genome builds and gene builds. Once the design is done the microarrays are often used for several years. The biologists working in EADGENE expressed the need of up-to-dated annotation files for the oligo-sets they share including information about the orthologous genes of model species, the Gene Ontology, the corresponding pathways and the chromosomal location.</p> <p>Results</p> <p>The results of SigReannot on a chicken micro-array used in the EADGENE project compared to the initial annotations show that 23% of the oligo-nucleotide gene annotations were not confirmed, 2% were modified and 1% were added. The interest of this up-to-date annotation procedure is demonstrated through the analysis of real data previously published.</p> <p>Conclusion</p> <p>SigReannot uses the oligo-nucleotide design procedure criteria to validate the probe-gene link and the Ensembl transcripts as reference for annotation. It therefore produces a high quality annotation based on reference gene sets.</p

Hepatic lipogenesis gene expression in two experimental egg-laying lines divergently selected on residual food consumption

Two Rhode Island Red egg-laying lines have been divergently selected on residual food intake (low intake R- line, high intake R+ line) for 19 generations. In addition to direct response, correlated responses have altered several other traits such as carcass adiposity and lipid contents of several tissues, the R+ animals being leaner than the R- ones. In a search for the biological origin of the differences observed in fat deposit, the hepatic mRNA amounts of genes involved in lipid metabolism were investigated. No difference was found between lines for mRNA levels of ATP citrate-lyase, acetyl-CoA carboxylase, fatty acid synthase, malic enzyme and CCAAT/enhancer binding protein α, a transcription factor acting on several lipogenesis genes. The genes coding for stearoyl-CoA desaturase and apolipoprotein A1 displayed significantly lower mRNA levels in the R+ cockerels compared to the R-. All together these mRNA levels explained 40% of the overall variability of abdominal adipose tissue weight, suggesting an important role of both genes in the fatness variability