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Plk4 Regulates Centriole Asymmetry and Spindle Orientation in Neural Stem Cells
Defects in mitotic spindle orientation (MSO) disrupt the organization of stem cell niches impacting tissue morphogenesis and homeostasis. Mutations in centrosome genes reduce MSO fidelity, leading to tissue dysplasia and causing several diseases such as microcephaly, dwarfism, and cancer. Whether these mutations perturb spindle orientation solely by affecting astral microtubule nucleation or whether centrosome proteins have more direct functions in regulatingMSO is unknown. To investigate this question, we analyzed the consequences of deregulating Plk4 (the master centriole duplication kinase) activity in Drosophila asymmetrically dividing neural stem cells. We found that Plk4 functions upstream of MSO control, orchestrating centriole symmetry breaking and consequently centrosome positioning. Mechanistically, we show that Plk4 acts through Spd2 phosphorylation, which induces centriole release from the apical cortex. Overall, this work not only reveals a role for Plk4 in regulating centrosome function but also links the centrosome biogenesis machinery with the MSO apparatus.ERC starting grant CentroStemCancer [242598]; Institut Curie; CNRS; NCI [P30CA23074]; NIGMS [R01 GM110166, GM126035]; FRM; IC; FRM installation grant; ATIP grantOpen access articleThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]
InstabilitĂ© gĂ©nĂ©tique : des dĂ©viations du contenu gĂ©nomique Ă la plasticitĂ© dâexpression des gĂšnes
Most animal cells are diploid, containing two copies of each chromosome. Establishment of proper bipolar mitotic spindle containing two centrosomes, one at each pole contributes to accurate chromosome segregation. This is essential for the maintenance of genome stability, tissue and organism homeostasis. However, numerical deviations to the diploid set are observed in healthy tissues. Polyploidy is the doubling of the whole chromosome set and aneuploidy concerns the gain or loss of whole chromosomes. Importantly, whole genome duplications and aneuploidy have also been associated to pathological conditions. For example, variations to genome content are associated with chromosome instability and cancer development, however their exact contribution to cancer genome remains poorly understood.In the first part of my PhD project, I investigated the consequences of polyploidy during cell division. I found that the presence of extra DNA and extra centrosomes generated invariably multipolar spindles. Then I identified contributors to the multipolar status using in vivo approaches in Drosophila neural stem cells and in vitro culture of cancer cells. Further I combined DNA and spindle perturbations with computer modelling and found that in polyploid cells, the presence of excessive DNA acts as a physical barrier blocking spindle pole coalescence and bipolarity. Indeed, laser ablation to disrupt and increase in microtubule stability and length to bypass the DNA-barrier could rescue bipolar spindle formation. This discovery challenges the current view that suggested extra-centrosomes as only contributor to spindle multipolarity and provides a rational to understand chromosome instability typical of polyploid cells.The aim of the second part of my PhD project was to generate a novel tool to quantitively probe chromosome loss in vivo in Drosophila tissues. Aneuploidy has been observed in various physiological tissues, however the frequency of this error remained highly debatable. In addition, tools developed so far to assess aneuploidy lack a temporal dimension. To circumvent this, I used the expression of a GFP report gene driven by the GAL4/UAS system and its inhibition by GAL80. In principle, the random loss of the chromosome carrying the GAL80 sequence leads to GFP appearance in aneuploid cells that can therefore be followed in live tissues. I found that chromosome loss was extremely infrequent in most tissues of the wild type fly. This tool combined with fluorescent marker and/or tested in various genetic background, might help understanding mechanisms behind aneuploidy genesis and outcome in vivo.While developing this tool, I discovered that in the larval brain, GFP cells where not a by-product of chromosome loss but rather an unexpected mis-regulation in the expression of the GAL80 gene. These results have strong implications for the Drosophila community as it can result in false positive in clonal experiments. Further, I discovered a mosaicism and plasticity of the Drosophila brain in neural stem cells for gene expression which differs from other organs and that is influenced by environmental stimuli. This possibly reflects a certain level of plasticity in the brain necessary for neuronal diversity, adaptation and survival.La plupart de nos cellules sont diploĂŻdes possĂ©dant deux copies de chaque chromosome. Lors de la mitose, la formation dâun fuseau bipolaire avec un centrosome Ă chaque pĂŽle permet la sĂ©grĂ©gation correcte des chromosomes, essentiel au maintien de la stabilitĂ© gĂ©nĂ©tique. Il existe nĂ©anmoins des variations du contenu chromosomique comme la polyploĂŻdie, dĂ©finit comme le doublement de lâensemble des chromosomes et lâaneuploĂŻdie, dĂ©finie comme la perte ou le gain de chromosome entier. Bien quâobservĂ©es, la frĂ©quence des cellules aneuploĂŻdies dans les tissus dâun organisme sain reste controversĂ©e.De façon importante, la duplication du gĂ©nome et lâaneuploĂŻdie sont associĂ©es Ă des pathologies et sont considĂ©rĂ©es comme une caractĂ©ristique du cancer. En effet, un nombre anormal de chromosomes est souvent associĂ© Ă une instabilitĂ© chromosomique. Toutefois le rĂŽle et les implications de ces variations dans lâinitiation et la progression de tumeur restent peu compris.Jâai dâabord Ă©tudiĂ© les consĂ©quences de la polyploĂŻdie sur la division des cellules. Jâai utilisĂ© des approches in vivo et in vitro en induisant la polyploidisation par dĂ©faut de cytocinĂšse dans des cellules souches neurales de drosophile et des cellules cancĂ©reuses humaines. Lâanalyse de leur mitose mâa permis de dĂ©couvrir que la prĂ©sence de chromosomes et de centrosomes en excĂšs conduisait invariablement Ă la formation de fuseaux multipolaires. En modĂ©lisant les cellules polyploĂŻdes, jâai dĂ©couvert quâau-delĂ de la quantitĂ©, la conformation spatiale de lâADN contribue Ă cette multipolaritĂ©. Des perturbations expĂ©rimentales au niveau de lâADN et du fuseau mâont permis de dĂ©montrer que la prĂ©sence dâADN en excĂšs agit comme une barriĂšre physique bloquant la coalescence des multiples pĂŽles et par consĂ©quent empĂȘchant la bipolaritĂ©. De façon intĂ©ressante, jâai rĂ©ussi Ă restaurer la bipolaritĂ© en supprimant la « barriĂšre dâADN » par ablation avec laser ou en augmentant la longueur des microtubules pour contourner celle-ci. Alors que lâamplification centrosomale Ă©tait considĂ©rĂ©e comme unique acteur, mes rĂ©sultats identifient lâexcĂšs dâADN comme contributeur clef de la multipolaritĂ© et de lâinstabilitĂ© chromosomique typique des cellules polyploĂŻdes.Puis, je me suis ensuite intĂ©ressĂ©e Ă lâaneuploĂŻdie, dont la frĂ©quence en contexte sain reste un sujet de dĂ©bat intense. De plus, les outils dĂ©veloppĂ©s jusquâĂ prĂ©sent pour Ă©valuer le taux dâaneuploĂŻdie manque dâune dimension temporelle. Jâai donc gĂ©nĂ©rĂ© un outil gĂ©nĂ©tique innovant de visualisation et de suivi des cellules aneuploĂŻdes in vivo chez la drosophile. Jâai utilisĂ© lâexpression de la GFP comme gĂšne rapporteur, contrĂŽlĂ©e par le systĂšme GAL4/UAS et son inhibition par GAL80. Ainsi, la perte alĂ©atoire du chromosome contenant la sĂ©quence du GAL80 entraine lâapparition dâun signal GFP dans les cellules aneuploĂŻdes. Celles-ci peuvent donc ĂȘtre facilement dĂ©tectĂ©es et suivies en temps-rĂ©el dans les tissus. Utilisant ce systĂšme, jâai dĂ©couvert que la perte de chromosome Ă©tait un Ă©vĂšnement trĂšs rare dans les tissus de la mouche. Cet outil combinĂ© Ă dâautres marqueurs fluorescents et/ou utilisĂ© dans divers contextes gĂ©nĂ©tiques pourrait aider Ă la comprĂ©hension de la genĂšse et du devenir des cellules aneuploĂŻdes in vivo.De plus, jâai constatĂ© que le cerveau de la larve prĂ©sentait un nombre important de cellules GFP. De maniĂšre surprenante, ces cellules ne rĂ©sultaient pas de la perte de chromosomes mais de la perte dâexpression du gĂšne GAL80. Ces rĂ©sultats inattendus ont de fortes implications pour la communautĂ© des drosophilistes car cela peut mener Ă des faux positifs dans les expĂ©riences de gĂ©nĂ©ration de clones. Jâai aussi dĂ©couvert que les cellules souches neurales prĂ©sentaient un mosaĂŻsme dans lâexpression des gĂšnes, qui diffĂšrent dâautres organes et sâadaptent Ă des stimuli environnementaux. Ceci reprĂ©sente possiblement un niveau de plasticitĂ© dans le cerveau nĂ©cessaire Ă la diversitĂ© neuronale, lâadaptation et la survie
Chromosomes function as a barrier to mitotic spindle bipolarity in polyploid cells
International audiencePloidy variations such as genome doubling are frequent in human tumors and have been associated with genetic instability favoring tumor progression. How polyploid cells deal with increased centrosome numbers and DNA content remains unknown. Using Drosophila neuroblasts and human cancer cells to study mitotic spindle assembly in polyploid cells, we found that most polyploid cells divide in a multipolar manner. We show that even if an initial centrosome clustering step can occur at mitotic entry, the establishment of kinetochore-microtubule attachments leads to spatial chromosome configurations, whereby the final coalescence of supernumerary poles into a bipolar array is inhibited. Using in silico approaches and various spindle and DNA perturbations, we show that chromosomes act as a physical barrier blocking spindle pole coalescence and bipolarity. Importantly, microtubule stabilization suppressed multipolarity by improving both centrosome clustering and pole coalescence. This work identifies inhibitors of bipolar division in polyploid cells and provides a rationale to understand chromosome instability typical of polyploid cancer cells