Meiosis in Plasmodium:How does it work?

Meiosis is sexual cell division, a process in eukaryotes whereby haploid gametes are produced. Compared to canonical model eukaryotes, meiosis in apicomplexan parasites appears to diverge from the process with respect to the molecular mechanisms involved; the biology of Plasmodium meiosis, and its regulation by means of post-translational modification, are largely unexplored. Here, we discuss the impact of technological advances in cell biology, evolutionary bioinformatics, and genome-wide functional studies on our understanding of meiosis in the Apicomplexa. These parasites, including Plasmodium falciparum, Toxoplasma gondii, and Eimeria spp., have significant socioeconomic impact on human and animal health. Understanding this key stage during the parasite's life cycle may well reveal attractive targets for therapeutic intervention.</p

Spatial organization of Rho GTPase signaling by RhoGEF/RhoGAP proteins

Rho GTPases control cell shape formation and thus fundamental physiological processes in all eukaryotes. Their functions are regulated by 145 RhoGEF and RhoGAP multi-domain proteins in humans. To provide the framework for a systems-level understanding of how these regulators orchestrate cellular morphogenesis, we comprehensively characterized their substrate specificities, localization and interactome. The resulting resource places the RhoGEFs/RhoGAPs in functional context, serving as a foundation for targeted and integrated studies. Our data reveals their critical role in the spatial organization of Rho signaling. They localize to multiple compartments to provide positional information, are extensively interconnected to jointly coordinate their signaling networks and are widely autoinhibited to remain sensitive to local activation. RhoGAPs exhibit lower substrate specificity than RhoGEFs and may contribute to preserving Rho activity gradients. We demonstrate the utility of our integrated data by detailing a multi-RhoGEF complex downstream of G-protein-coupled receptors in which the enzymes mutually regulate their activities

IDENTIFICATION AND ANALYSIS OF A NOVEL ROLE FOR THE TOUSLED-LIKE KINASE IN REGULATING MITOTIC SPINDLE DYNAMICS

Deregulation of kinase activity is one example of how cells become cancerous by evading evolutionary constraints. The Tousled kinase (Tsl) was initially identified in Arabidopsis thaliana as a developmentally important kinase. There are two mammalian orthologues of Tsl and one orthologue in C. elegans, TLK-1, which is essential for embryonic viability and germ cell development. Depletion of TLK-1 leads to embryonic arrest large, distended nuclei, and ultimately embryonic lethality. Prior to terminal arrest, TLK-1-depleted embryos undergo aberrant mitoses characterized by poor metaphase chromosome alignment, delayed mitotic progression, lagging chromosomes, and supernumerary centrosomes. I discovered an unanticipated requirement for TLK-1 in mitotic spindle assembly and positioning. Normally, in the newly-fertilized zygote (P0) the maternal pronucleus migrates toward the paternal pronucleus at the posterior end of the embryo. After pronuclear meeting, the pronuclear-centrosome complex rotates 90° during centration to align on the anteroposterior axis followed by nuclear envelope breakdown (NEBD). However, in TLK-1-depleted P0 embryos, the centrosome-pronuclear complex rotation is significantly delayed with respect to NEBD and chromosome congression, Additionally, centrosome positions over time in tlk-1(RNAi) early embryos revealed a defect in posterior centrosome positioning during spindle-pronuclear centration, and 4D analysis of centrosome positions and movement in newly fertilized embryos showed aberrant centrosome dynamics in TLK-1-depleted embryos. Several mechanisms contribute to spindle rotation, one of which is the anchoring of astral microtubules to the cell cortex. Attachment of these microtubules to the cortices is thought to confer the necessary stability and forces in order to rotate the centrosome-pronuclear complex in a timely fashion. Analysis of a microtubule end-binding protein revealed that TLK-1-depleted embryos exhibit a more stochastic distribution of microtubule growth toward the cell cortices, and the types of microtubule attachments appear to differ from wild-type embryos. Additionally, fewer astral microtubules are in the vicinity of the cell cortex, thus suggesting that the delayed spindle rotation could be in part due to a lack of appropriate microtubule attachments to the cell cortex. Together with recently published biochemical data revealing the Tousled-like kinases associate with components of the dynein microtubule motor complex in humans, these data suggest that Tousled-like kinases play an important role in mitotic spindle assembly and positioning

YEAST HASPIN KINASE REGULATES MITOTIC CELL CYCLE EVENTS: FROM G2/M TRANSITION TO POLARISOME DISPERSION

Haspin is a serine/threonine atypical kinase that phosphorylates histone H3-T3 during metaphase, promoting the recruitment of the chromosomal passenger complex (CPC) at kinetochores. Haspin depletion leads to cell arrest in mitosis and prevents proper chromosome positioning at the metaphase plate. Saccharomyces cerevisiae genome encodes for two haspin paralogues ALK1 and ALK2. We recently showed that these genes are essential to coordinate polarization and cell cycle progression, ensuring the correct positioning of several polarity factors following a transient mitotic delay. The aim of this project is to identify new processes where haspin kinase is involved. The first part of this work shows that Alk1 has a role at the G2/M transition in S. cerevisiae. These findings constitute the first evidence for Alk1-specific functions that are not shared by its paralogue Alk2. Our results indicate that cells lacking ALK1 are sensitive to Latrunculin A and complete nuclear division within the unbudded mother cells. These observations pointed toward a defect in the morphogenesis checkpoint. We also observed that in absence of ALK1 the Cdc28-Y19 phosphorylation signal decreases significantly during a morphogenetic stress. Exploring the underlying mechanism, we found that the decrease in phosphorylation is caused by a misregulation in Mih1 phosphatase activity in absence of Alk1. Therefore in budding yeast Alk1 modulates G2/M cell cycle switch by regulating Mih1 3 activity. The second part of this work is focused on exploring the role of Alk1 and Alk2 in polarisome dispersion. We show that the previously reported role of haspin in polarization relies on its ability to modulate Ras localization. Our observations are indicative for a mitotic role of Ras, which, by regulating Cdc24 redistribution, influences Cdc42 activation at polarized sites. These observations may help to shed light on alterations in cell polarity, which often constitute the molecular mechanism for cancer insurgence

Identification of a myotubularin-related phosphatase that regulates autophagic flux and lysosome homeostasis

Macroautophagy (autophagy) is a vesicle trafficking process that targets cytoplasmic cargoes to the lysosome for degradation and underlies multiple human disorders. Pioneering work in Saccharomyces cerevisiae defined the core autophagy machinery, but animals possess autophagy regulators that were not identified in yeast. Autophagic flux occurs when autophagy rate increases or decreases in response to various cellular cues, such as nutrient availability. Indeed, dysregulated autophagy rates contribute to disease, making autophagy- modulation a therapeutic avenue to treat cancer, neurodegenerative disorders, and other diseases. To identify novel regulators of autophagy in animals, I investigated autophagy in the context of animal development using Drosophila. In my dissertation, I screened for phosphoinositide phosphatases that influence autophagy, and identifed CG3530/dMtmr6, a previously uncharacterized phosphatase. CG3530/dMtmr6 is homologous to the human MTMR6 subfamily of myotubularin-related 3-phosphoinositide phosphatases. I showed that dMtmr6 functions as a regulator of autophagic flux in multiple Drosophila cell types, and the MTMR6 family member MTMR8 functions similarly in autophagy of higher animal cells. Decreased dMtmr6 function resulted in autophagic vesicle accumulation, lysosome biogenesis, and impaired both fluid phase endocytosis in the fat body and phagocytosis in embryonic macrophages. Additionally, dMtmr6 is required for development and viability in Drosophila. In human cells, lysosome homeostasis requires both the MTMR8 PH domain and catalytic cysteine residue, but only the PH domain is required to maintain autophagic flux. Collectively, this work identified a role for dMtmr6 and MTMR8 in autophagic flux and lysosome homeostasis

An Asymmetrically Balanced Organization of Kinases versus Phosphatases across Eukaryotes Determines Their Distinct Impacts

<div><p>Protein phosphorylation underlies cellular response pathways across eukaryotes and is governed by the opposing actions of phosphorylating kinases and de-phosphorylating phosphatases. While kinases and phosphatases have been extensively studied, their organization and the mechanisms by which they balance each other are not well understood. To address these questions we performed quantitative analyses of large-scale 'omics' datasets from yeast, fly, plant, mouse and human. We uncovered an asymmetric balance of a previously-hidden scale: Each organism contained many different kinase genes, and these were balanced by a small set of highly abundant phosphatase proteins. Kinases were much more responsive to perturbations at the gene and protein levels. In addition, kinases had diverse scales of phenotypic impact when manipulated. Phosphatases, in contrast, were stable, highly robust and flatly organized, with rather uniform impact downstream. We validated aspects of this organization experimentally in nematode, and supported additional aspects by theoretic analysis of the dynamics of protein phosphorylation. Our analyses explain the empirical bias in the protein phosphorylation field toward characterization and therapeutic targeting of kinases at the expense of phosphatases. We show quantitatively and broadly that this is not only a historical bias, but stems from wide-ranging differences in their organization and impact. The asymmetric balance between these opposing regulators of protein phosphorylation is also common to opposing regulators of two other post-translational modification systems, suggesting its fundamental value.</p></div

The influence of cell size on cytokinesis in situ and genomic interrogation of human cell size regulation

La cellule est l’élément fondamental de la vie. Plus d’une vingtaine de trillions de cellules forment les organes et tissus de notre corps. Ces cellules sont de taille spécifique puisqu’elles ont des fonctions précises au sein de leur tissu respectif. Dans la plupart des cas, les cellules doivent proliférer en se divisant pour se renouveler et ainsi assurer le bon fonctionnement d’un organisme. La dernière étape de la division cellulaire, la cytokinèse, est exécutée par la contraction d’un anneau contractile d’actomyosine, nécessaire pour effectuer la séparation physique de la cellule en deux cellules filles. La première partie des travaux décrits dans cet ouvrage portent sur la caractérisation de la cytokinèse en utilisant, comme modèle in vivo, les cellules précurseur de la vulve (VPCs) du nématode C. elegans. Notre étude révèle que plusieurs aspects de l’anneau d’actomyosine s’ajustent en fonction de la taille de la cellule. Entre autres, la largeur de l’anneau contractile, juste avant sa constriction, s’ajuste en fonction de la longueur des VPCs. De plus, la rapidité avec laquelle l’anneau se contracte dépend de la circonférence de la cellule. Ces découvertes nous ont amené à nous demander comment la cellule régule sa taille? Les cellules en prolifération maintiennent leur taille en homéostasie en équilibrant leur taux de croissance et de division cellulaire. Afin d’interroger les gènes impliqués dans le maintien de la taille cellulaire du mammifère, nous avons utilisé la technologie CRISPR/Cas9, afin d’éliminer par délétion tous les gènes humains, à raison d’un par cellule, pour identifier ceux qui causent une augmentation ou une diminution de la taille cellulaire. Cette étude nous a permis d’identifier plusieurs gènes déjà connus régulant la croissance cellulaire. De plus, nous avons identifié un groupe de gènes, incluant TLE4 un corépresseur de la transcription que nous avons caractérisé, n’ayant jamais été associé avec une fonction de contrôle de la taille cellulaire chez les mammifères. En somme, nos travaux ont contribué à l’approfondissement des connaissances sur la division cellulaire, plus précisément la cytokinèse, et des gènes impliqués dans le maintien de la taille cellulaire. Une meilleure connaissance du fonctionnement de ces deux évènements cellulaires est essentielle puisque leur dérégulation peut entrainer plusieurs pathologies, incluant le cancer.Cells are the fundamental building blocks of life. The human body contains over twenty trillion cells that make up the different tissues and organs of our bodies. Cells within organs are of specific sizes to perform their specialized functions. In most cases, these cells must divide to proliferate and replenish the population of cells essential for proper organism function. The final stage of cellular division, termed cytokinesis, entails the assembly and constriction of a contractile ring that drives the dramatic cell shape changes required to physically partition the cell into two daughter cells. The first part of the work presented in this thesis addresses the characterization of cytokinesis in the epithelial vulval precursor cells (VPCs) of the nematode worm C. elegans. This study principally revealed that several aspects of cytokinesis scale with cell size. For instance, I observed that the breadth of the actomyosin ring scaled with VPC length. In addition, the speed of contractile ring constriction scaled with the circumference of VPCs. These scaling events raised the more general question as to how cells regulate their size. Proliferating cells attain cell size homeostasis by balancing cell growth and cell division. In order to define the molecular regulators of size in human cells a genome-wide approach was taken. Recently developed CRISPR/Cas9 technology was used to perform the first pooled knockout screens for human cell size regulators in the NALM-6 pre-B lymphocytic cell line. These screens revealed many genes that affect the size of NALM-6 cells, a number of which were previously known to be involved in growth regulation. In addition, these screens revealed the identity of many genes with no previously established functions associated with cell size regulation. Amongst the previously unknown regulators, I characterized the function of a co-repressor of transcription, TLE4, which I showed functions as a regulator of the B-cell lineage. This work contributes to the knowledge of the mechanics of cytokinesis in C. elegans epithelial cells and of the genes that coordinate cell size in humans. These results provide insights into cell growth and division in normal cells and how these processes may be perturbed in cancer and other diseases

Bcl-xL (S49) and (S62) sequential phosphorylation/dephosphorylation during mitosis prevents chromosome instability and aneuploidy

Une caractéristique intéressante de la protéine Bcl-xL est la présence d'un domaine en boucle non-structurée entre les hélices α1 and α2 de la protéine. Ce domaine protéique n'est pas essentiel pour sa fonction anti-apoptotique et absent chez CED-9, la protéine orthologue chez Caenorhabditis elegans. A l'intérieur de ce domaine, Bcl-xL subit une phosphorylation et déphosphorylation dynamique sur les résidus Ser49 et Ser62 en phase G2 du cycle cellulaire et lors de la mitose. Lorsque ces résidus sont mutés et les protéines exprimées dans des cellules cancéreuses, les cellules démontrent plusieurs défauts mitotiques liés à l'instabilité chromosomique. Pour analyser les effets de Bcl-xL Ser49 et Ser62 dans les cellules normales, les présentes études ont été réalisées dans des cellules diploïdes humaines normales, et in vivo chez Caenorhabditis elegans. Dans une première étude, nous avons utilisé la lignée cellulaire de cellules fibroblastiques diploïdes humaines normales BJ, exprimant Bcl-xL (type sauvage), (S49A), (S49D), (S62A), (S62D) et les double (S49/62A) et (S49/62D) mutants. Les cellules exprimant les mutants de phosphorylation ont montré des cinétiques de doublement de la population cellulaire réduites. Ces effets sur la cinétique de doublement de la population cellulaire corrèle avec l'apparition de la sénescence cellulaire, sans impact sur les taux de mort cellulaire. Ces cellules sénescentes affichent des phénotypes typiques de sénescence associés notamment à haut niveau de l'activité β-galactosidase associée à la sénescence, la sécrétion d' interleukine-6, l'activation de p53 et de p21WAF1/ Cip1, un inhibiteur des complexes kinase cycline-dépendant, ainsi que la formation de foyers de chromatine nucléaire associés à γH2A.X. Les analyses de fluorescence par hybridation in situ et des caryotypes par coloration au Giemsa ont révélé que l'expression des mutants de phosphorylation de Bcl-xL provoquent de l'instabilité chromosomique et l'aneuploïdie. Ces résultats suggèrent que les cycles de phosphorylation et déphosphorylation dynamiques de Bcl-xL Ser49 et Ser62 sont importants dans le maintien de l'intégrité des chromosomes lors de la mitose dans les cellules normales. Dans une deuxième étude, nous avons entrepris des expériences chez Caenorhabditis elegans pour comprendre l'importance des résidus Ser49 et Ser62 de Bcl-xL in vivo. Les vers transgéniques portant les mutations de Bcl-xL (S49A, S62A, S49D, S62D et S49/62A) ont été générés et leurs effets ont été analysés sur les cellules germinales des jeunes vers adultes. Les vers portant les mutations de Bcl-xL ont montré une diminution de ponte et d'éclosion des oeufs, des variations de la longueur de leurs régions mitotiques et des zones de transition, des anomalies chromosomiques à leur stade de diplotène, et une augmentation de l'apoptose des cellules germinales. Certaines de ces souches transgéniques, en particulier les variants Ser/Ala, ont également montré des variations de durée de vie par rapport aux vers témoins. Ces observations in vivo ont confirmé l'importance de Ser49 et Ser62 à l'intérieur du domaine à boucle de Bcl-xL pour le maintien de la stabilité chromosomique. Ces études auront une incidence sur les futures stratégies visant à développer et à identifier des composés qui pourraient cibler non seulement le domaine anti-apoptotique de la protéine Bcl-xL, mais aussi son domaine mitotique pour la thérapie du cancer.An interesting feature of Bcl-xL protein is the presence of an unstructured loop domain between its α1 and α2 helices, a domain not essential for its anti-apoptotic function and absent in CED-9, ortholog protein in Caenorhabditis elegans. Within this domain, Bcl-xL undergoes dynamic phosphorylation and dephosphorylation at Ser49 and Ser62 during G2 and mitosis in human cancer cells. When these residues are mutated and proteins expressed in cancer cells, cells harbor mitotic defects, including chromosome mis-attachment, lagging, bridging and mis-segregation, events associated with chromosome instability and aneuploidy. To further analyze the effects of Bcl-xL Ser49 and Ser62 in normal cells, the present studies were performed in normal human diploid cells, and in vivo in Caenorhabditis elegans. First, we studied normal human diploid BJ foreskin fibroblast cells expressing Bcl-xL(wild type), (S49A), (S49D), (S62A), (S62D) and the dual (S49/62A) and (S49/62D) mutants. Cells expressing S49 and/or S62 phosphorylation mutants showed reduced kinetics of cell population doubling. These effects on cell population doubling kinetics correlated with early outbreak of senescence with no impact on the cell death rate. Senescent cells displayed typical senescence-associated phenotypes including high-level of senescence-associated β-galactosidase activity, interleukin-6 secretion, tumor suppressor p53 and cyclin-dependent kinase inhibitor p21Waf1/Cip1 activation as well as γH2A.X-associated nuclear chromatin foci. Fluorescence in situ hybridization analysis and Giemsa-banded karyotypes revealed that the expression of Bcl-xL phosphorylation mutants in normal diploid BJ cells provoked chromosome instability and aneuploidy. These findings suggest that dynamic Bcl-xL Ser49 and Ser62 phosphorylation/ dephosphorylation cycles are important in the maintenance of chromosome integrity during mitosis in normal cells. Second, we undertook experiments in Caenorhabditis elegans to understand the importance of Bcl-xL Ser49 and Ser62 in vivo. Transgenic worms carrying single-site S49A, S62A, S49D, S62D and dual-site S49/62A mutants were generated and their effects were analyzed in germlines of young adult worms. Worms expressing Bcl-xL variants showed decreased egg-laying and hatching, variations in the length of their mitotic regions and transition zones, chromosomal abnormalities at their diplotene stages, and increased germline apoptosis. Some of these transgenic strains, particularly the Ser to Ala variants, also showed slight modulations of lifespan compared to their controls. The in vivo observations confirmed the importance of Ser49 and Ser62 within the loop domain of Bcl-xL in maintaining chromosome stability. These studies could impact future strategies aiming to develop and identify compounds that could target not only the anti-apoptotic domain of Bcl-xL protein, but also its mitotic domain for cancer therapy