Rôles et régulation du PI(4,5)P2 dans le remodelage cortical et la morphogénèse cellulaire en mitose

La division cellulaire doit être précisément contrôlée dans le temps et dans l'espace pour permettre la formation de deux cellules filles, au contenu génétique identique à celui de la cellule mère. Ceci requiert des modifications successives de la forme des cellules, induites par un remodelage du cortex cellulaire. A ce jour, les mécanismes moléculaires du contrôle de l'organisation corticale restent mal caractérisés. Nos travaux chez la drosophile mettent en évidence que le PI(4,5)P2, un phosphoinositide de la membrane plasmique, se concentre à l'équateur des cellules dés l'entrée en anaphase. Ce PI(4,5)P2 est nécessaire à la stabilité corticale des cellules en mitose, via l'activation de la dMoésine. Celle-ci, en liant l'Actine à la membrane plasmique, joue un rôle clé dans l'organisation du cortex des cellules mitotiques et dans la régulation de ses propriétés mécaniques. Nous montrons que l'interaction PI(4,5)P2/dMoésine participe à la contraction cellulaire à l'entrée en mitose, puis à l'élongation cellulaire caractéristique des étapes plus tardives. En fin de mitose, la phosphatase Pp1-87B inactive la dMoésine. Par un crible fonctionnel systématique, nous avons mis en évidence le rôle majeur de deux voies de biosynthèse qui agissent en synergie pour produire localement du PI(4,5)P2, dépendantes de Skittles et de Pten, ainsi que le rôle d'une troisième voie nécessitant l'activité de la protéine dOcrl pour contrôler l'homéostasie du PI(4,5)P2. Ensemble, ces résultats permettent de mieux comprendre les mécanismes qui contrôlent le remodelage cortical et les modifications de forme cellulaire qui ont lieu en mitose.Cell division must be accurately controlled in time and space to permit the formation of two daughter cells whose genetic content is identical to that of the mother cell. This process requires successive modifications of cell shape, induced by cortical remodelling. Molecular mechanisms controlling cortical reorganization during mitosis remain partially uncharacterized. Our work in Drosophila cells demonstrates that PI(4,5)P2, a phosophoinositide of the plasma membrane, is enriched at the equatorial plate at the onset of anaphase. This PI(4,5)P2 is necessary for the cortical stability of mitotic cells and requires dMoesin activation. The dMoesin, linking actin to the plasma membrane, plays a critical role in the cortical organization of mitotic cells and in the regulation of its mechanical properties. We show that the interaction PI(4,5)P2/dMoesin participates in cellular contraction at the beginning of mitosis, then in cell elongation characteristic of subsequent steps. At the end of mitosis, the Pp1-87B phosphatase inactivates the dMoesin. By a systematic functional screen, we characterize the key role of two pathways acting in synergy to locally produce PI(4,5)P2, Skittles- and Pten-dependent, and the role of a third pathway requiring dOcrl activity to control PI(4,5)P2 homeostasis. Altogether, these results allow us to better understand the mechanisms controlling cortical remodelling and modifications of cell shape that occur during mitosis

Spatio-temporally separated cortical flows and spindle geometry establish physical asymmetry in fly neural stem cells

Asymmetric cell division, creating sibling cells with distinct developmental potentials, can be manifested in sibling cell size asymmetry. This form of physical asymmetry occurs in several metazoan cells, but the underlying mechanisms and function are incompletely understood. Here we use Drosophila neural stem cells to elucidate the mechanisms involved in physical asymmetry establishment. We show that Myosin relocalizes to the cleavage furrow via two distinct cortical Myosin flows: at anaphase onset, a polarity induced, basally directed Myosin flow clears Myosin from the apical cortex. Subsequently, mitotic spindle cues establish a Myosin gradient at the lateral neuroblast cortex, necessary to trigger an apically directed flow, removing Actomyosin from the basal cortex. On the basis of the data presented here, we propose that spatiotemporally controlled Myosin flows in conjunction with spindle positioning and spindle asymmetry are key determinants for correct cleavage furrow placement and cortical expansion, thereby establishing physical asymmetry

Engineered kinases as a tool for phosphorylation of selected targets in vivo

Reversible protein phosphorylation by kinases controls a plethora of processes essential for the proper development and homeostasis of multicellular organisms. One main obstacle in studying the role of a defined kinase-substrate interaction is that kinases form complex signaling networks and most often phosphorylate multiple substrates involved in various cellular processes. In recent years, several new approaches have been developed to control the activity of a given kinase. However, most of them fail to regulate a single protein target, likely hiding the effect of a unique kinase-substrate interaction by pleiotropic effects. To overcome this limitation, we have created protein binder-based engineered kinases that permit a direct, robust, and tissue-specific phosphorylation of fluorescent fusion proteins in vivo. We show the detailed characterization of two engineered kinases based on Rho-associated protein kinase (ROCK) and Src. Expression of synthetic kinases in the developing fly embryo resulted in phosphorylation of their respective GFP-fusion targets, providing for the first time a means to direct the phosphorylation to a chosen and tagged target in vivo. We presume that after careful optimization, the novel approach we describe here can be adapted to other kinases and targets in various eukaryotic genetic systems to regulate specific downstream effectors

Molecular networks linked by Moesin drive remodeling of the cell cortex during mitosis

During mitosis, cortical Moesin activity is restricted to promote cell elongation and cytokinesis, but localized Moesin recruitment is necessary for polar bleb retraction and cortical relaxation

Control of asymmetric cell division

Asymmetric cell division (ACD) is a mechanism to generate cellular diversity and used by prokaryotes and eukaryotes alike. Stem cells in particular rely on ACD to self-renew the stem cell while simultaneously generating a differentiating sibling. It is well established that the differential partitioning of cell fate determinants in the form of RNA and proteins between sibling cells induces changes in cell behavior and fate. Recently, insight into molecular mechanisms has been gained that could explain how centrosomes and centrosome-associated structures such as histones, chromosomes or the primary cilium, segregate asymmetrically. Similarly, many cell types also generate physical asymmetry in the form of sibling cell size differences. Emerging data suggests that spindle-induced cleavage furrow positioning through regulated spindle placement and spindle geometry is insufficient to explain all occurrence of cell-size asymmetry. Instead, asymmetric membrane extension based on asymmetric Myosin localization and cortical remodeling could be a driving force for the generation of physical asymmetry

Rôles et régulation du PI(4,5)P2 dans le remodelage cortical et la morphogénèse cellulaire en mitose

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Polarized SCAR and the Arp2/3 complex regulate apical cortical remodeling in asymmetrically dividing neuroblasts

Summary: Although the formin-nucleated actomyosin cortex has been shown to drive the changes in cell shape that accompany animal cell division in both symmetric and asymmetric cell divisions, the mitotic role of cortical Arp2/3-nucleated actin networks remain unclear. Here using asymmetrically dividing Drosophila neural stem cells as a model system, we identify a pool of membrane protrusions that form at the apical cortex of neuroblasts as they enter mitosis. Strikingly, these apically localized protrusions are enriched in SCAR, and depend on SCAR and Arp2/3 complexes for their formation. Because compromising SCAR or the Arp2/3 complex delays the apical clearance of Myosin II at the onset of anaphase and induces cortical instability at cytokinesis, these data point to a role for an apical branched actin filament network in fine-tuning the actomyosin cortex to enable the precise control of cell shape changes during an asymmetric cell division

Asymmetrically dividing Drosophila neuroblasts utilize two spatially and temporally independent cytokinesis pathways

Precise cleavage furrow positioning is required for faithful chromosome segregation and cell fate determinant distribution. In most metazoan cells, contractile ring placement is regulated by the mitotic spindle through the centralspindlin complex, and potentially also the chromosomal passenger complex (CPC). Drosophila neuroblasts, asymmetrically dividing neural stem cells, but also other cells utilize both spindle-dependent and spindle-independent cleavage furrow positioning pathways. However, the relative contribution of each pathway towards cytokinesis is currently unclear. Here we report that in Drosophila neuroblasts, the mitotic spindle, but not polarity cues, controls the localization of the CPC component Survivin. We also show that Survivin and the mitotic spindle are required to stabilize the position of the cleavage furrow in late anaphase and to complete furrow constriction. These results support the model that two spatially and temporally separate pathways control different key aspects during asymmetric cell division, ensuring correct cell fate determinant segregation and neuroblast self-renewal

Asymmetric chromatin retention and nuclear envelopes separate chromosomes in fused cells in vivo.

Hybrid cells derived through fertilization or somatic cell fusion recognize and separate chromosomes of different origins. The underlying mechanisms are unknown but could prevent aneuploidy and tumor formation. Here, we acutely induce fusion between Drosophila neural stem cells (neuroblasts; NBs) and differentiating ganglion mother cells (GMCs) in vivo to define how epigenetically distinct chromatin is recognized and segregated. We find that NB-GMC hybrid cells align both endogenous (neuroblast-origin) and ectopic (GMC-origin) chromosomes at the metaphase plate through centrosome derived dual-spindles. Physical separation of endogenous and ectopic chromatin is achieved through asymmetric, microtubule-dependent chromatin retention in interphase and physical boundaries imposed by nuclear envelopes. The chromatin separation mechanisms described here could apply to the first zygotic division in insects, arthropods, and vertebrates or potentially inform biased chromatid segregation in stem cells

Multiplex lateral flow assay for rapid visual blood group genotyping

International audienceConventional blood group phenotyping by hemagglutination assays, carried out pretransfusion, is unsuitable in certain clinical situations. Molecular typing offers an alternative method, allowing the deduction of blood group phenotype from genotype. However, current methods require a long turnaround time and are not performed on-site, limiting their application in emergency situations. Here, we report the development of a novel, rapid multiplex molecular method to identify seven alleles in three clinically relevant blood group systems (Kidd, Duffy and MNS). Our test, using a dry-reagent allele-specific lateral flow biosensor, does not require DNA extraction and allows easy visual determination of blood group genotype. Multiplex linear-after-the-exponential (LATE)-PCR and lateral flow parameters were optimized with a total processing time of one hour from receiving the blood sample. Our assay had a 100 % concordance rate between the deduced and the standard serological phenotype in a sample from 108 blood donors, showing the accuracy of the test. Owing to its simple handling, the assay can be operated by non-skilled health-care professionals. The proposed assay offers the potential for the development of other relevant single nucleotide polymorphism (SNP) panels for immunohematology and new applications, such as for infectious diseases, in the near future