Phosphatidylinositol bisphosphate (PIP2) directly regulates electrophysiological activity in a diverse family of ion channels whether the effect is stimulatory or inhibitory. Much has been unveiled about the apparent affinity and modulatory function of PIP2 using a chemically modified dioctanoyl PIP2 (diC8), a membrane delimited cytosolic co-factor in inside-out macropatch experiments. Yet, the scarcity of molecular tools that permit fine external control in whole-cell systems has precluded future studies from probing the physiological role of PIP2 in cells in the presence of a fully intact cytoplasm. Here we introduce light as an external control for PIP2 through photocaging of diC8, and test its activation of Kir2.3 (IRK3), an inwardly rectifying ion channel that has previously shown to possess moderate binding affinity to PIP2, in excised, inside-out macropatches. Our experiments revealed that photocaged-diC8 and irradiated photocaged-diC8 have significantly different activation kinetics than the fully active diC8. Surprisingly, the activation of caged-diC8 by UV irradiation attenuated Kir2.3 activity, while the inactivated diC8 (caged-diC8) resulted in similar magnitude of channel activity compared to the currents elicited by unmodified diC8. Interestingly, we also show that application of both activated (irradiated) and inactive (caged) diC8 in macropatches generated highly fluctuating ion channel activity.

Cytokinesis, the final step of cell division, is characterized by formation of a cleavage furrow that ingresses to separate the cell into two daughter cells. This process requires rearrangement of the cytoskeleton and addition of membrane to the growing furrow. The phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] has been implicated in regulating both actin dynamics and membrane trafficking and, thus, is uniquely poised to coordinate these different processes during cytokinesis. In this study, I show that PtdIns(4,5)P2 is involved in another aspect of cytokinesis: regulation of actomyosin contractility. Perturbing PtdIns(4,5)P2 levels in Drosophila spermatocytes caused constriction to fail and cleavage furrows to regress. Moreover, PtdIns(4,5)P2 hydrolysis is implicated in this process: inhibiting PLC or IP3R or chelating Ca2+ also caused defects in furrow ingression. In addition, I show that PLC and MLCK activities are required for myosin light chain phosphorylation and for proper myosin and actin localization to the cleavage furrow. Thus, I propose a model in which PtdIns(4,5)P2 hydrolysis-dependent Ca2+ release activates MLCK via Ca2+/calmodulin to maintain myosin filaments in the contractile ring and regulate cleavage furrow ingression. Furthermore, I show that PtdIns(4,5)P2 has a role in maintaining contractile ring components in the cleavage furrow that does not depend on PtdIns(4,5)P2 hydrolysis. I conclude that PtdIns(4,5)P2 regulates myosin contractility through a PLC-dependent pathway leading to myosin phosphorylation and is also involved in localizing contractile ring components to the furrow. Thus, PtdIns(4,5)P2 may coordinate signals leading to cytoskeleton rearrangement and actomyosin contractility during cytokinesis.

Cytokinesis, the final step of cell division, is characterized by formation of a cleavage furrow that ingresses to separate the cell into two daughter cells. This process requires rearrangement of the cytoskeleton and addition of membrane to the growing furrow. The phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] has been implicated in regulating both actin dynamics and membrane trafficking and, thus, is uniquely poised to coordinate these different processes during cytokinesis. In this study, I show that PtdIns(4,5)P2 is involved in another aspect of cytokinesis: regulation of actomyosin contractility. Perturbing PtdIns(4,5)P2 levels in Drosophila spermatocytes caused constriction to fail and cleavage furrows to regress. Moreover, PtdIns(4,5)P2 hydrolysis is implicated in this process: inhibiting PLC or IP3R or chelating Ca2+ also caused defects in furrow ingression. In addition, I show that PLC and MLCK activities are required for myosin light chain phosphorylation and for proper myosin and actin localization to the cleavage furrow. Thus, I propose a model in which PtdIns(4,5)P2 hydrolysis-dependent Ca2+ release activates MLCK via Ca2+/calmodulin to maintain myosin filaments in the contractile ring and regulate cleavage furrow ingression. Furthermore, I show that PtdIns(4,5)P2 has a role in maintaining contractile ring components in the cleavage furrow that does not depend on PtdIns(4,5)P2 hydrolysis. I conclude that PtdIns(4,5)P2 regulates myosin contractility through a PLC-dependent pathway leading to myosin phosphorylation and is also involved in localizing contractile ring components to the furrow. Thus, PtdIns(4,5)P2 may coordinate signals leading to cytoskeleton rearrangement and actomyosin contractility during cytokinesis.

L'activation des lymphocytes T est un événement fondamental de la réponse immunitaire adaptative. Elle est déclenchée par la transduction du signal médiée par le complexe TCR/CD3.Le mécanisme de déclenchement du signal via le TCR reste, mal compris. Mon projet de thèse vise à examiner la contribution des PI(4,5)P2 dans le mécanisme du déclenchement du signal TCR. L'expression ectopique d'une phosphatase spécifique de PIP2 a permis de réduire de 50% les PI(4,5)P2 membranaires. L'analyse du profil de phosphorylations spécifiques des tyrosines montre que l'expression ectopique de cette 5-phosphatase augmente les événements de phosphorylation à l'état basal comparés à des conditions analogues pour des cellules contrôles. En revanche, alors que suite à l'engagement du TCR par le complexe MHC-peptide indépendamment du co-récepteur CD4 nous observons une augmentation des phosphorylations, l'activation par le complexe MHC-peptide CD4 dépendant semble affectée. Nous avons analysé la contribution des PI(4,5)P2 dans la dynamique membranaire du TCR grâce à la technique svFCS. PIP2 peuvent jouer un rôle essentiel dans la régulation de la dynamique latérale du TCR à la membrane plasmique. Enfin, nous avons observé que l'inhibition par la néomycine (aminoglycoside qui en tant que polycation peut se lier et neutraliser le groupement anionique de PI(4,5)P2), aboutit à des changements similaires dans la dynamique membranaire du TCR et la régulation proximale dans des cellules T primaires murines CD4+. Ensemble, nos données révèlent le rôle régulateur fondamental de PI(4,5)P2 dans la dynamique membranaire du TCR et de CD4, pour le contrôle de l'initiation des voies de signalisation du TCR. / PI(4,5)P2 plays important roles in a large spectrum of membrane-based cellular activities . It is therefore surprising that it is currently not known if PI(4,5)P2 is also involved in the T cell receptor (TCR) signal transduction mechanism. We investigate here the role of PI(4,5)P2 in the regulation of the TCR membrane dynamics and signaling initiation using a combination of biophysical, biochemistry and cell biology approaches. Ectopic expression of the Inp54p, a 5-phophatase that hydrolyzes PI(4,5)P2 into PI(4)P, with a membrane targeting signal specifically decreased by 50% of the PI(4,5)P2 in a CD4+ T cell hybridoma. Interestingly, we observed that this decrease caused modified TCR (and CD4 co-receptor) dynamics in the plasma membrane. The lateral diffusion switched from a regime dominated by dynamic partitioning in the cholesterol- and sphingolipid-dependent nanodomains into one dominated by dynamic partitioning in the actin cytoskeleton-assisted nanodomains. This switch was associated with a change in activation of the TCR and proximal signaling pathways both at the basal level and upon stimulation. Upon pMHC engagement, the CD4-independent activation of the TCR signaling pathways was found significantly augmented while that of CD4-dependent was affected. We further provided evidence for the involvement of PI(4,5)P2 in the Finally, we found that inhibition of interactions between PI(4,5)P2 and endogenous proteins with neomycin resulted in the modified TCR membrane dynamics and proximal signaling in primary murine CD4+ T cells. Altogether, our data reveal that PI(4,5)P2 is crucially involved in the control of the activation of TCR early signaling pathways.

Inwardly rectifying potassium (Kir) channels are critically important for regulating resting membrane potential in excitable cells, a job underscored by the severe pathophysiology associated with channel dysfunction. While all Kir channels require the activating lipid PIP2, many of these channels have diverse modulatory factors that couple to PIP2-dependent gating. Channels in the Kir3 (GIRK) family, in particular, have several co-activating elements, including G-protein betagamma subunits, ethanol, and sodium. During stimulation of Gq-coupled receptors, downstream activation of Protein Kinase C can phosphorylate and inhibit Kir3 channels, yet the mechanism of inhibition and phosphorylation sites are incompletely understood. We took a combined experimental and computational approach using neuronal Kir3.2 to investigate how phosphorylation at a putative PKC site identified in Kir3.1/3.4 could lead to channel inhibition. Kir3.2 inhibition was found to depend on the phosphorylation state of Ser-196, although mutagenesis data suggest it functions as an allosteric regulator of PKC inhibition. MD simulations identified a molecular switch whereby phosphorylation of Ser-196 recruits a critical gating residue, Arg-201, away from the sodium coordination site Asp-228. Neutralization of Ser-196 or Arg-201 resulted in less active channels which exhibited increased sensitivity to PKC inhibition. Additionally the interplay of PIP2 and PKC inhibition was examined in depth using homomeric Kir3.2, revealing that increases in channel-PIP2 interactions limit sensitivity to PKC inhibition, whereas low levels of PIP2 increase PKC sensitivity. Neutralization of Ser-196 uncoupled PKC inhibition from this PIP2 dependence. These studies suggest a model whereby PKC inhibition can occur along PIP2-dependent and PIP2-independent pathways, depending on the phosphorylation state of Ser-196.

Human MYO1C gene encodes three myosin 1c (Myo1c) isoforms which differ only at their N-ends. Interestingly, all three isoforms localize to the nucleus and also to the cytoplasm, where they are anchored to the plasma membrane by the interaction with phosphatidyl inositol-4,5-bisphosphate (PIP2). However, studies reporting functional involvement of these isoforms are inconsistent. While the shortest isoform C (Myo1c-isoC) has been implicated exclusively in the cytoplasmic processes, the longer isoform B (termed the nuclear myosin 1, NM1) has been employed in the nuclear and processes, such as DNA transcription and rRNA maturation. Similarly, the longest isoform A (Myo1c-isoA) exerts its functions in the nucleus solely. To complete the information on the cellular functions of Myo1c isoforms, we searched for the cytoplasmic functions of NM1 and nuclear functions of Myo1c-isoC. In mouse, only two isoforms (NM1 and Myo1c-isoC) are expressed. We prepared the knock-out mouse (KO) which lacks specifically NM1 while retaining Myo1c-isoC unchanged. Surprisingly, this manifested in no phenotype observed. Since we demonstrated that even Myo1c-isoC acts in the transcription in the similar manner as NM1, it suggests that Myo1c- isoC functionally overlap with NM1 in the nuclear functions. Besides its localization...

Les membres de la famille des 5-phosphatases d’inositols polyphosphates et de phosphoinositides sont des enzymes caractérisées par la présence de deux domaines catalytiques conservés qui hydrolysent un phosphate en position 5 sur un noyau inositol. SKIP (Skeletal Muscle and Kidney enriched Inositol Phosphatase), également appelée Pps (Putative PI 5-phosphatase) est un des derniers membres de la famille des 5-phosphatases à avoir été découvert à ce jour. Cette enzyme hydrolyse majoritairement le phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) et le phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3). Les phosphoinositides (PtdIns) représentent environ 10% des lipides membranaires et sont impliqués dans de nombreuses cascades de signalisation cellulaire conduisant, entre autres, à la prolifération, l’apoptose, la différenciation, la sécrétion, le trafic vésiculaire et la mobilité cellulaire. Des études de surexpression de SKIP en cellules tendent à montrer que cette protéine pourrait jouer un rôle de régulateur négatif dans la formation du cytosquelette d’actine et/ou dans la voie de signalisation de l’insuline. Afin d’étudier in vivo la fonction de la protéine SKIP chez la souris, nous avons décidé de générer des souris transgéniques surexprimant cette protéine de manière conditionnelle. Dans ce but, nous avons infecté des embryons murins par des lentivirus porteurs d’un transgène SKIP et avons obtenu, après réimplantation des embryons infectés dans des femelles pseudogestantes, deux lignées de souris transgéniques. Celles-ci ont ensuite été croisées avec des souris exprimant la recombinase Cre de manière ubiquitaire afin de pouvoir activer la transcription de SKIP dans l’ensemble des organes. Des expériences de Western blot, de dosage d’activité 5-phosphatase ainsi que des PCR en temps réel sont venus confirmer la présence de la protéine transgénique et de son activité catalytique. L’ensemble des expériences qui ont été menées du point de vue phénotypique tend à montrer que dans notre modèle, la surexpression de SKIP ne provoque aucune anomalie évidente du point de vue anatomique, glycémique ou immunologique. Toutefois, des expériences concernant la physiologie rénale ont été réalisées sur base des résultats d’immunohistochimie et nous ont permis de détecter une anomalie dans les mécanismes de réabsorption d’eau ainsi que dans l’expression et la phosphorylation des canaux hydriques AQP2.

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