Dimeric and tetrameric forms of muscle fructose-1,6- bisphosphatase play different roles in the cell

Muscle fructose 1,6-bisphosphatase (FBP2), besides being a regulatory enzyme of glyconeogenesis also protects mitochondria against calcium stress and plays a key role in regulation of the cell cycle, promoting cardiomyocytes survival. However, in cancer cells, FBP2 acts as an anti-oncogenic/anti-proliferative protein. Here, we show that the physiological function of FBP2 depends both on its level of expression in a cell as well as its oligomerization state. Animal fructose-1,6-bisphosphatases are thought to function as tetramers. We present evidence that FBP2 exists in an equilibrium between tetramers and dimers. The dimeric form is fully active and insensitive to AMP, the main allosteric inhibitor of FBP2. Tetramerization induces the sensitivity of the protein to AMP, but it requires the presence of a hydrophobic central region in which leucine 190 plays a crucial role. Only the tetrameric form of FBP2 is retained in cardiomyocyte cell nucleus whereas only the dimeric form associates with mitochondria and protects them against stress stimuli, such as elevated calcium and H2O2 level. Remarkably, in hypoxic conditions, which are typical for many cancers, FBP2 ceases to interact with mitochondria and loses its pro-survival potential. Our results throw new light on the basis of the diverse role of FBP2 in cells

Architecture and permeability of post-cytokinesis plasmodesmata lacking cytoplasmic sleeves

This work was supported by the grants by the Region Aquitaine (to E.M.B) and PEPS (Initial Support for Exploratory Projects to E.M.B) and National Agency for Research (Grant ANR-14-CE19-0006-01 to E.M.B).Plasmodesmata are remarkable cellular machines responsible for the controlled exchange of proteins, small RNAs and signalling molecules between cells. They are lined by the plasma membrane (PM), contain a strand of tubular endoplasmic reticulum (ER), and the space between these two membranes is thought to control plasmodesmata permeability. Here, we have reconstructed plasmodesmata three-dimensional (3D) ultrastructure with an unprecedented level of 3D information using electron tomography. We show that within plasmodesmata, ER-PM contact sites undergo substantial remodelling events during cell differentiation. Instead of being open pores, post-cytokinesis plasmodesmata present such intimate ER-PM contact along the entire length of the pores that no intermembrane gap is visible. Later on, during cell expansion, the plasmodesmata pore widens and the two membranes separate, leaving a cytosolic sleeve spanned by tethers whose presence correlates with the appearance of the intermembrane gap. Surprisingly, the post-cytokinesis plasmodesmata allow diffusion of macromolecules despite the apparent lack of an open cytoplasmic sleeve, forcing the reassessment of the mechanisms that control plant cell-cell communication.PostprintPeer reviewe

Bloom’s Syndrome and PICH Helicases Cooperate with Topoisomerase IIα in Centromere Disjunction before Anaphase

Centromeres are specialized chromosome domains that control chromosome segregation during mitosis, but little is known about the mechanisms underlying the maintenance of their integrity. Centromeric ultrafine anaphase bridges are physiological DNA structures thought to contain unresolved DNA catenations between the centromeres separating during anaphase. BLM and PICH helicases colocalize at these ultrafine anaphase bridges and promote their resolution. As PICH is detectable at centromeres from prometaphase onwards, we hypothesized that BLM might also be located at centromeres and that the two proteins might cooperate to resolve DNA catenations before the onset of anaphase. Using immunofluorescence analyses, we demonstrated the recruitment of BLM to centromeres from G2 phase to mitosis. With a combination of fluorescence in situ hybridization, electron microscopy, RNA interference, chromosome spreads and chromatin immunoprecipitation, we showed that both BLM-deficient and PICH-deficient prometaphase cells displayed changes in centromere structure. These cells also had a higher frequency of centromeric non disjunction in the absence of cohesin, suggesting the persistence of catenations. Both proteins were required for the correct recruitment to the centromere of active topoisomerase IIα, an enzyme specialized in the catenation/decatenation process. These observations reveal the existence of a functional relationship between BLM, PICH and topoisomerase IIα in the centromere decatenation process. They indicate that the higher frequency of centromeric ultrafine anaphase bridges in BLM-deficient cells and in cells treated with topoisomerase IIα inhibitors is probably due not only to unresolved physiological ultrafine anaphase bridges, but also to newly formed ultrafine anaphase bridges. We suggest that BLM and PICH cooperate in rendering centromeric catenates accessible to topoisomerase IIα, thereby facilitating correct centromere disjunction and preventing the formation of supernumerary centromeric ultrafine anaphase bridges

The impact of cyclin-dependent kinase 5 depletion on poly(ADP-ribose) polymerase activity and responses to radiation

Cyclin-dependent kinase 5 (Cdk5) has been identified as a determinant of sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. Here, the consequences of its depletion on cell survival, PARP activity, the recruitment of base excision repair (BER) proteins to DNA damage sites, and overall DNA single-strand break (SSB) repair were investigated using isogenic HeLa stably depleted (KD) and Control cell lines. Synthetic lethality achieved by disrupting PARP activity in Cdk5-deficient cells was confirmed, and the Cdk5KD cells were also found to be sensitive to the killing effects of ionizing radiation (IR) but not methyl methanesulfonate or neocarzinostatin. The recruitment profiles of GFP-PARP-1 and XRCC1-YFP to sites of micro-irradiated Cdk5KD cells were slower and reached lower maximum values, while the profile of GFP-PCNA recruitment was faster and attained higher maximum values compared to Control cells. Higher basal, IR, and hydrogen peroxide-induced polymer levels were observed in Cdk5KD compared to Control cells. Recruitment of GFP-PARP-1 in which serines 782, 785, and 786, potential Cdk5 phosphorylation targets, were mutated to alanines in micro-irradiated Control cells was also reduced. We hypothesize that Cdk5-dependent PARP-1 phosphorylation on one or more of these serines results in an attenuation of its ribosylating activity facilitating persistence at DNA damage sites. Despite these deficiencies, Cdk5KD cells are able to effectively repair SSBs probably via the long patch BER pathway, suggesting that the enhanced radiation sensitivity of Cdk5KD cells is due to a role of Cdk5 in other pathways or the altered polymer levels

CD56 is a pathogen recognition receptor on human natural killer cells

Aspergillus (A.) fumigatus is an opportunistic fungal mold inducing invasive aspergillosis (IA) in immunocompromised patients. Although antifungal activity of human natural killer (NK) cells was shown in previous studies, the underlying cellular mechanisms and pathogen recognition receptors (PRRs) are still unknown. Using flow cytometry we were able to show that the fluorescence positivity of the surface receptor CD56 significantly decreased upon fungal contact. To visualize the interaction site of NK cells and A. fumigatus we used SEM, CLSM and dSTORM techniques, which clearly demonstrated that NK cells directly interact with A. fumigatus via CD56 and that CD56 is re-organized and accumulated at this interaction site time-dependently. The inhibition of the cytoskeleton showed that the receptor re-organization was an active process dependent on actin re-arrangements. Furthermore, we could show that CD56 plays a role in the fungus mediated NK cell activation, since blocking of CD56 surface receptor reduced fungal mediated NK cell activation and reduced cytokine secretion. These results confirmed the direct interaction of NK cells and A. fumigatus, leading to the conclusion that CD56 is a pathogen recognition receptor. These findings give new insights into the functional role of CD56 in the pathogen recognition during the innate immune response

Exopolysaccharide-Independent Social Motility of Myxococcus xanthus

Social motility (S motility), the coordinated movement of large cell groups on agar surfaces, of Myxococcus xanthus requires type IV pili (TFP) and exopolysaccharides (EPS). Previous models proposed that this behavior, which only occurred within cell groups, requires cycles of TFP extension and retraction triggered by the close interaction of TFP with EPS. However, the curious observation that M. xanthus can perform TFP-dependent motility at a single-cell level when placed onto polystyrene surfaces in a highly viscous medium containing 1% methylcellulose indicated that “S motility” is not limited to group movements. In an apparent further challenge of the previous findings for S motility, mutants defective in EPS production were found to perform TFP-dependent motility on polystyrene surface in methylcellulose-containing medium. By exploring the interactions between pilin and surface materials, we found that the binding of TFP onto polystyrene surfaces eliminated the requirement for EPS in EPS- cells and thus enabled TFP-dependent motility on a single cell level. However, the presence of a general anchoring surface in a viscous environment could not substitute for the role of cell surface EPS in group movement. Furthermore, EPS was found to serve as a self-produced anchoring substrate that can be shed onto surfaces to enable cells to conduct TFP-dependent motility regardless of surface properties. These results suggested that in certain environments, such as in methylcellulose solution, the cells could bypass the need for EPS to anchor their TPF and conduct single-cell S motility to promote exploratory movement of colonies over new specific surfaces

Quelle fonction pour la CLIP-170 ? Recherche de partenaires et nouveaux outils d'investigation.

CLIP-170 links endosomes and microtubules (MTs) in vitro. It also treadmills at the +ends of MTs and associates with kinetochores and MT cortical anchors. Its behaviour at MT +ends is that of a +TIPs, of which EB1 is a member. +TIPs recruit molecular complexes at MT +ends to regulate MT dynamics and to mediate cargo loading or interactions with cortical cues. Here we demonstrate interaction between CLIP-170 and LIS1 via its distal zinc fingers. Our results indicate that LIS1 recruitment to kinetochores is dynein/dynactin dependent, and that recruitment there of CLIP-170 is dependent on its binding site to LIS1. Overexpression of CLIP-170 results in a zinc finger dependent localization of phospho-LIS1 and dynactin to MT bundles, raising the possibility that the latter is targeted via phospho-LIS1 and dynein. This work suggests that LIS1 is a regulated adaptor between CLIP-170 and cytoplasmic dynein at sites involved in cargo-MT loading, and/or in control of MT dynamics. We are using expression of siRNA and microinjection of region specific CLIP-170 antibodies, to investigate its functions. A Ct-pAb or a Nt-pAb displaced it from both MT plus ends in interphase and KTs in mitosis. With both pAb, EB1 remained localised to MT +ends while p150Glued was released into the cytoplasm. In addition, p150Glued was still localised at unattached KTs. Same results were obtained when CLIP-170 expression level was dramatically diminished by siRNA expression. CLIP-170 antibody microinjection did not dramatically affect mitosis, as determined on fixed cells. These preliminary findings support our model that D/D is addressed to the +TIPs through its interaction with CLIP-170 via LIS1. We are now evaluating by nC 3D videomicroscopy the effects of siRNA expression and microinjection on the interphase MT dynamics and on the poleward sliding of KTs to the mitotic spindle.Le terme de CLIP-170 désigne la protéine de lien cytoplasmique de 170 KDa, isolée par Rickard et Kreis (1990). In vitro, elle constitue un lien statique entre les endosomes et les microtubules (MTs). En chacun des points où la CLIP-170 est localisée, elle se co-distribue avec le complexe moteur dynéine/dynactine (D/D). Les auteurs ont initialement établi un modèle de fonctionnement de concert de ces trois protagonistes : la CLIP-170 établirait le lien initial entre le cargo et le MT. Le complexe D/D serait recruté sur le cargo. Une fois le moteur associé au MT, le lien statique serait levé, rendant possible le mouvement. Ce modèle offrait une explication aux données d'immunolocalisation. Toutefois, le fonctionnement de concert de la CLIP-170 et du moteur moléculaire nécessitait que l'on puisse trouver une interaction entre les protagonistes. Les travaux présentés dans cette thèse apportent pour la première fois la preuve d'une interaction indirecte entre le complexe D/D et la CLIP-170. LIS1 est une protéine codée par le gène causal du syndrome de Miller-Dieker, une forme de lissencéphalie de type I. Elle sert d'adaptateur entre le domaine carboxy-terminal de la CLIP-170 et le complexe moteur dont elle régule l'activité. Nos résultats établissent que le domaine d'interaction de la CLIP-170 avec LIS1 est requis pour l'adressage de la première aux kinétochores prémétaphasiques. Il est nécessaire à l'adressage de LIS1 (et du complexe moteur) aux bouts (+) des MTs interphasiques. Nous présentons deux nouvelles approches permettant d'interférer avec le fonctionnement de la CLIP-170 tant en interphase qu'en mitose : la microinjection d'anticorps dirigés contre les domaines extrêmes de la protéine, ainsi que l'utilisation de siRNA dirigés contre l'ARNm la codant. Combinées aux techniques de suivi de protéines fluorescentes par vidéomicroscopie 3D développées au laboratoire, elles permettront d'interférer avec le fonctionnement de la CLIP-170 et de définir ainsi son rôle