Analyse et modélisation de la dynamique des chromosomes au cours de la ségrégation mitotique dans la levure à fission

La ségrégation correcte des chromosomes est un élément essentiel dans le contrôle de la stabilité génomique chez les eucaryotes. Des défauts de ségrégation conduisent à l'apparition de cellules filles aneuploïdes (ie : nombre incorrect de chromosomes), pouvant ainsi être la cause des avortements spontanés ou entraîner des maladies génétiques comme la trisomie 21. L'aneuploïdie est fréquemment observée dans les tumeurs humaines et joue un rôle clef dans le développement et/ou la progression des cancers. Des études récentes suggèrent que les défauts d'attachements des chromosomes seraient la cause majeure des erreurs de ségrégation et de l'aneuploïdie. Les kinétochores (Kt), complexes multiprotéiques situés sur chaque chromosome répliqué, interagissent avec des microtubules (MTs) provenant de pôles opposés ce qui permettra leur séparation en anaphase. Lorsque tous les chromosomes sont bi-orientés, les protéines des points de contrôle mitotiques (Mad2, Bub1, Mad1 etc.) partent du kinétochore permettant la dégradation des cohésines, les chromosomes se séparent alors. Si un chromosome n’est pas correctement attaché, les protéines des points de contrôle persistent sur le Kt, empêchant la dégradation de la cohésine, on parle alors d'activation du point de contrôle. Très récemment, il a été montré que c’est la tension appliquée par les MTs sur le Kt (tension intra-kinetochorienne) qui est responsable de la levée du point de contrôle mitotique. Les acteurs impliqués dans l'établissement de cette tension sont inconnus. La dynéine a été impliquée dans la levée du point de contrôle chez les eucaryotes supérieurs. Dans la levure à fission, nous avons montré qu'elle participe à la dynamique des chromosomes, à la stabilité génétique et que sa délétion provoque une activation du point de contrôle mitotique sans empêcher son inactivation (article 1). Des résultats préliminaires suggèrent aussi que la dynéine est impliquée dans la correction des attachements mérotéliques (lorsqu’un kinétochore est attaché aux deux pôles). L'attachement mérotélique jouerait un rôle clé dans la mise en place de l'instabilité chromosomique. Pour aller plus loin dans l'étude du rôle de la dynéine, nous avons caractérisé précisément l'impact de la mérotélie sur la progression mitotique (article 2). Nous avons pu établir que l'attachement mérotélique était corrigé par un mécanisme dépendant de la tension en anaphase B. Pour mieux comprendre les mécanismes de mise sous tension des kinétochores en mitose, nous nous sommes intéressés au rôle du complexe Dam1. Nous avons démontré son rôle dans la dynamique des microtubules en interphase et dans le mouvement de retour des kinétochores en anaphase A (article 3). L'ensemble de ce travail illustre la complexité des mécanismes conduisant à l'attachement correct des chromosomes aux microtubules, processus fondamental pour maintenir la stabilité génomique.The correct segregation of chromosomes is an essential element in the control of genomic stability in eukaryotes. Segregation defects lead to the appearance of aneuploid daughter cells (ie : incorrect number of chromosomes) and this process may well be the cause of spontaneous abortions or genetic disorders such as trisomy 21. Aneuploidy is frequently observed in human tumours and plays a key role in the development and / or progression of cancer. Recent studies suggest that defects in chromosome attachment are the major cause of aneuploidy. Kinetochores (Kt), multiprotein complexes located on each replicated chromosome, interact with microtubules (MTs) from opposite poles, which allow their separation in anaphase. When all chromosomes are bi-oriented, proteins of the mitotic checkpoint (Mad2, Bub1, Mad1 etc..) leave the kinetochore, degradation of cohesin takes place, and chromosomes separate. If a chromosome is not properly attached, the checkpoint proteins persist on Kt, preventing the degradation of cohesin. Very recently, it was shown that it is the tension applied by the Kt-MTs, which is responsible for the removal of the mitotic checkpoint proteins. The actors involved in the establishment of this tension are unknown. Dynein has been implicated in the checkpoint inhibition in higher eukaryotes. In fission yeast, we showed that dynein participates to chromosome dynamics, genetic stability and that dynein deletion causes activation of the mitotic checkpoint without preventing its inactivation (Section 1). Preliminary results also suggest that dynein is involved in correcting merotelic attachments (when a kinetochore is attached to both poles). Merotelic attachment plays a key role in the development of chromosomal instability. To go further in studying the role of dynein, we characterized the precise impact of merotely on mitotic progression (Article 2). We were able to establish that merotelic attachment is corrected by a tension-dependent mechanism in anaphase B. To better understand the mechanisms of tension applied on kinetochores during mitosis, we investigated the role of the Dam1 complex. We demonstrated that Dam1 plays a key role in controlling microtubule dynamics in interphase and that it also controls kinetochore poleward movement in anaphase A (Section 3). This work illustrates the complexity of the mechanisms leading to the correct attachment of chromosomes to microtubules, a process that is fundamental to maintain genomic stability

Ase1/Prc1-dependent spindle elongation corrects merotely during anaphase in fission yeast

The tug of war that ensues when a kinetochore binds microtubules from both spindle poles is resolved by Ase1/Prc1

First person - Thibault Courtheoux and Alghassimou Diallo

International audienceFirst Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Thibault Courtheoux and Alghassimou Diallo are joint first authors on 'Aurora A kinase activity is required to maintain an active spindle assembly checkpoint during pro-metaphase', published in Journal of Cell Science. Thibault is a postdoc and Alghassimou a PhD student in the lab of Dr Claude Prigent at IGDR-UMR, Universite de Rennes, France, investigating cell division and mechanisms leading to chromosome instability and cancer

Aurora A kinase activity is required to maintain an active spindle assembly checkpoint during prometaphase

International audienceDuring the prometaphase stage of mitosis, the cell builds a bipolar spindle of microtubules that mechanically segregates sister chromatids between two daughter cells in anaphase. The spindle assembly checkpoint (SAC) is a quality control mechanism that monitors proper attachment of microtubules to chromosome kinetochores during prometaphase. Segregation occurs only when each chromosome is bi-oriented with each kinetochore pair attached to microtubules emanating from opposite spindle poles. Overexpression of the protein kinase Aurora A is a feature of various cancers and is thought to enable tumour cells to bypass the SAC, leading to aneuploidy. Here, we took advantage of a chemical and chemical-genetic approach to specifically inhibit Aurora A kinase activity in late prometaphase. We observed that a loss of Aurora A activity directly affects SAC function, that Aurora A is essential for maintaining the checkpoint protein Mad2 on unattached kinetochores and that inhibition of Aurora A leads to loss of the SAC, even in the presence of nocodazole or Taxol. This is a new finding that should affect the way Aurora A inhibitors are used in cancer treatments.This article has an associated First Person interview with the first authors of the paper

Cortical dynamics during cell motility are regulated by CRL3 KLHL21 E3 ubiquitin ligase

Directed cell movement involves spatial and temporal regulation of the cortical microtubule (Mt) and actin networks to allow focal adhesions (FAs) to assemble at the cell front and disassemble at the rear. Mts are known to associate with FAs, but the mechanisms coordinating their dynamic interactions remain unknown. Here we show that the CRL3KLHL21 E3 ubiquitin ligase promotes cell migration by controlling Mt and FA dynamics at the cell cortex. Indeed, KLHL21 localizes to FA structures preferentially at the leading edge, and in complex with Cul3, ubiquitylates EB1 within its microtubule-interacting CH-domain. Cells lacking CRL3KLHL21 activity or expressing a non-ubiquitylatable EB1 mutant protein are unable to migrate and exhibit strong defects in FA dynamics, lamellipodia formation and cortical plasticity. Our study thus reveals an important mechanism to regulate cortical dynamics during cell migration that involves ubiquitylation of EB1 at focal adhesions.ISSN:2041-172

Mitotic redistribution of the mitochondrial network by Miro and Cenp-F

Although chromosome partitioning during mitosis is well studied, the molecular mechanisms that allow proper segregation of cytoplasmic organelles in human cells are poorly understood. Here we show that mitochondria interact with growing microtubule tips and are transported towards the daughter cell periphery at the end of mitosis. This phenomenon is promoted by the direct and cell cycle-dependent interaction of the mitochondrial protein Miro and the cytoskeletal-associated protein Cenp-F. Cenp-F is recruited to mitochondria by Miro at the time of cytokinesis and associates with microtubule growing tips. Cells devoid of Cenp-F or Miro show decreased spreading of the mitochondrial network as well as cytokinesis-specific defects in mitochondrial transport towards the cell periphery. Thus, Miro and Cenp-F promote anterograde mitochondrial movement and proper mitochondrial distribution in daughter cells.ISSN:2041-172

Aurora-A phosphorylates splicing factors and regulates alternative splicing

ABSTRACT Aurora-A kinase is well known to regulate progression through mitosis. However, the kinase also performs additional functions that could explain the failure of its inhibitors to be effective in cancer treatments. To identify these functions, we applied a proteomics approach to search for interactors of Aurora-A. We found a large number of proteins involved in pre-mRNA splicing, strongly suggesting an important role for Aurora-A in this biological process. Consistently, we first report the subcellular localization of Aurora-A in nuclear speckles, the storehouse of splicing proteins. We also demonstrate direct interaction of Aurora-A with RRM domain-containing splicing factors such as hnRNP and SR proteins and their phosphorylation in vitro . Further, RNA-sequencing analysis following pharmacological inhibition of Aurora-A resulted in alternative splicing changes corresponding to 505 genes, including genes with functions regulated by Aurora-A kinase. Finally, we report enrichment of RNA motifs within the alternatively spliced regions affected by Aurora-A kinase inhibition which are bound by Aurora-A interacting splicing factors, suggesting that Aurora-A regulates alternative splicing by modulating the activity of these interacting splicing factors. Overall our work identified Aurora-A as a novel splicing kinase and for the first time, describes a broad role of Aurora-A in regulating alternative splicing

A cullin-RING ubiquitin ligase targets exogenous α-synuclein and inhibits Lewy body–like pathology

Parkinson’s disease (PD) is a neurological disorder characterized by the progressive accumulation of neuronal α-synuclein (αSyn) inclusions called Lewy bodies. It is believed that Lewy bodies spread throughout the nervous system due to the cell-to-cell propagation of αSyn via cycles of secretion and uptake. Here, we investigated the internalization and intracellular accumulation of exogenous αSyn, two key steps of Lewy body pathogenesis, amplification and spreading. We found that stable αSyn fibrils substantially accumulate in different cell lines upon internalization, whereas αSyn monomers, oligomers, and dissociable fibrils do not. Our data indicate that the uptake-mediated accumulation of αSyn in a human-derived neuroblastoma cell line triggered an adaptive response that involved proteins linked to ubiquitin ligases of the S-phase kinase-associated protein 1 (SKP1), cullin-1 (Cul1), and F-box domain–containing protein (SCF) family. We found that SKP1, Cul1, and the F-box/LRR repeat protein 5 (FBXL5) colocalized and physically interacted with internalized αSyn in cultured cells. Moreover, the SCF containing the F-box protein FBXL5 (SCFFBXL5) catalyzed αSyn ubiquitination in reconstitution experiments in vitro using recombinant proteins and in cultured cells. In the human brain, SKP1 and Cul1 were recruited into Lewy bodies from brainstem and neocortex of patients with PD and related neurological disorders. In both transgenic and nontransgenic mice, intracerebral administration of exogenous αSyn fibrils triggered a Lewy body–like pathology, which was amplified by SKP1 or FBXL5 loss of function. Our data thus indicate that SCFFXBL5 regulates αSyn in vivo and that SCF ligases may constitute targets for the treatment of PD and other α-synucleinopathies

A stochastic model of kinetochore–microtubule attachment accurately describes fission yeast chromosome segregation

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