Structural centrosome aberrations promote non-cell-autonomous invasiveness

Centrosomes are the main microtubule-organizing centers of animal cells. Although centrosome aberrations are common in tumors, their consequences remain subject to debate. Here, we studied the impact of structural centrosome aberrations, induced by deregulated expression of ninein-like protein (NLP), on epithelial spheres grown in Matrigel matrices. We demonstrate that NLP-induced structural centrosome aberrations trigger the escape (“budding”) of living cells from epithelia. Remarkably, all cells disseminating into the matrix were undergoing mitosis. This invasive behavior reflects a novel mechanism that depends on the acquisition of two distinct properties. First, NLP-induced centrosome aberrations trigger a re-organization of the cytoskeleton, which stabilizes microtubules and weakens E-cadherin junctions during mitosis. Second, atomic force microscopy reveals that cells harboring these centrosome aberrations display increased stiffness. As a consequence, mitotic cells are pushed out of mosaic epithelia, particularly if they lack centrosome aberrations. We conclude that centrosome aberrations can trigger cell dissemination through a novel, non-cell-autonomous mechanism, raising the prospect that centrosome aberrations contribute to the dissemination of metastatic cells harboring normal centrosomes

Vers une approche comportementale de distinction des phases d'apprentissage procédural : le cas d'une procédure d'assemblage en environnement virtuel

National audienceVirtual environments used to acquire procedures and transfer them to real-life situations often lack adaptability to the learning rhythm of individual learners. The aim of the present study was to analyze the progress of procedural learning and to examine the relevance of a behavioral indicator to automatically identify procedural learning phases in real time. Identifying the progression of each learner would enable adapting teaching scenarios. Sixty-three individuals participated in the experiment to learn a Soma cube assembly procedure in a virtual environment, based on graphical instructions. Analysis of the activity traces and mental workload showed a progression in performance and a reduction in cognitive cost as the task was practiced. A proposal for dividing learners' activity into phases is analyzed, and prospects for improvement are provided.Les environnements virtuels exploités pour acquérir des procédures et les transférer à des situations réelles manquent souvent d'adaptabilité face au rythme d'apprentissage de chaque apprenant. L'objectif de la présente étude était d'analyser le déroulement de l'apprentissage de procédure et d'examiner la pertinence d'un indicateur comportemental pour repérer en temps réel et de façon automatique les phases d'apprentissage procédural. L'identification de la progression de chaque apprenant permettrait d'adapter les scénarios pédagogiques. Soixante-trois individus ont participé à l'expérience afin d'apprendre une procédure d'assemblage du cube de Soma en environnement virtuel à partir d'instructions graphiques. L'analyse des traces de l'activité ainsi que de la charge de travail mental ont montré une progression des performances et une diminution du coût cognitif au fur et à mesure de la pratique de la tâche. Une proposition de découpage de l'activité des apprenants par phases est analysée et des perspectives d'amélioration sont fournies

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Movie 4 related to fig2c: Basal extrusion of GFP-CEP131 expressing cell from MDCK 2D monolayer. from Structural centrosome aberrations sensitize polarized epithelia to basal cell extrusion

Movie shows brightfield signal (upper movie) and corresponding fluorescence signals (lower movie) representing mCardinal-ZO-1 (red) and GFP-CEP131 (green) (lower movie). MDCK were cells cultured in 2D monolayer, induced to express GFP-CEP131 for 48 hours and treated with etoposide to stimulate cell extrusion. Images were acquired every 20 minutes and movie displays 1 frame per second. Scale bars represent 10 microns

Vers l'identification des phases d'apprentissage procédural en environnement virtuel

National audienceVirtual environments used to acquire skills generate large amounts of behavioral data and thus provide new opportunities to understand learners' activity. From such data, a clustering method was used in the present study to identify phases in the procedural learning process. The recognition of these phases from a behavioral point of view would allow to follow in real time and in an automatic way the progress of the individuals and thus to personalize the pedagogical scenarios in a virtual environment. Sixty-three participants repeatedly performed a Soma cube assembly procedure in a virtual environment. Analyses show an improvement in performance and a decrease in cognitive cost as the task is repeated. The results of a multidimensional approach of dividing the activity into different phases are presented and discussed.Les environnements virtuels utilisés pour acquérir des compétences génèrent de grandes quantités de données comportementales et apportent ainsi de nouvelles opportunités de compréhension de l'activité des apprenants. A par-tir de telles données, une méthode de clustering a été utilisée dans la présente étude afin d'identifier des phases dans le processus d'apprentissage procédural. La reconnaissance de ces phases d'un point de vue comportemental permettrait de suivre en temps réel et de façon automatique la progression des individus et ainsi personnaliser les scénarios pédagogiques en environnement virtuel. Soixante-trois participants ont réalisé de façon répétée une procédure d'assemblage du cube de Soma en environnement virtuel. Les analyses montrent une amélioration des performances et une diminution du coût cognitif au fur et à mesure des répétitions de la tâche. Les résultats d'une approche multidimensionnelle de découpage de l'activité en différentes phases sont présentés et discutés

Flow cytometry to sort mammalian cells in cytokinesis.

International audienceBACKGROUND: Cell division or cytokinesis, which results from a series of events starting in metaphase, is the mechanism by which the mother cell cytoplasm is divided between the two daughter cells. Hence it is the final step of the cell division cycle. The aim of the present study was to demonstrate that mammalian cells undergoing cytokinesis can be sorted selectively by flow cytometry. MATERIALS AND METHODS: Cultures of HeLa cells were arrested in prometaphase by nocodazole, collected by mitotic shake-off and released for 90 min into fresh medium to enrich for cells undergoing cytokinesis. After ethanol fixation and DNA staining, cells were sorted based on DNA content and DNA fluorescence signal height. RESULTS: We define a cell population that transiently accumulates when synchronized cells exit mitosis before their entry into G1. We show that this population is highly enriched in cells undergoing cytokinesis. In addition, this population of cells can be sorted and analyzed by immunofluorescence and western blotting. CONCLUSIONS: This method of cell synchronization and sorting provides a simple means to isolate and biochemically analyze cells in cytokinesis, a period of the cell cycle that has been difficult to study by cell fractionation

Synergic reprogramming of mammalian cells by combined exposure to mitotic Xenopus egg extracts and transcription factors

Transfer of somatic cell nuclei to enucleated eggs and ectopic expression of specific transcription factors are two different reprogramming strategies used to generate pluripotent cells from differentiated cells. However, these methods are poorly efficient, and other unknown factors might be required to increase their success rate. Here we show that Xenopus egg extracts at the metaphase stage (M phase) have a strong reprogramming activity on mouse embryonic fibroblasts (MEFs). First, they reset replication properties of MEF nuclei toward a replication profile characteristic of early development, and they erase several epigenetic marks, such as trimethylation of H3K9, H3K4, and H4K20. Second, when MEFs are reversibly permeabilized in the presence of M-phase Xenopus egg extracts, they show a transient increase in cell proliferation, form colonies, and start to express specific pluripotency markers. Finally, transient exposure of MEF nuclei to M-phase Xenopus egg extracts increases the success of nuclear transfer to enucleated mouse oocytes and strongly synergizes with the production of pluripotent stem cells by ectopic expression of transcription factors. The mitotic stage of the egg extract is crucial, because none of these effects is detected when using interphasic Xenopus egg extracts. Our data demonstrate that mitosis is essential to make mammalian somatic nuclei prone to reprogramming and that, surprisingly, the heterologous Xenopus system has features that are conserved enough to remodel mammalian nuclei

Genome-scale analysis of metazoan replication origins reveals their organization in specific but flexible sites defined by conserved features

In metazoans, thousands of DNA replication origins (Oris) are activated at each cell cycle. Their genomic organization and their genetic nature remain elusive. Here, we characterized Oris by nascent strand (NS) purification and a genome-wide analysis in Drosophila and mouse cells. We show that in both species most CpG islands (CGI) contain Oris, although methylation is nearly absent in Drosophila, indicating that this epigenetic mark is not crucial for defining the activated origin. Initiation of DNA synthesis starts at the borders of CGI, resulting in a striking bimodal distribution of NS, suggestive of a dual initiation event. Oris contain a unique nucleotide skew around NS peaks, characterized by G/T and C/A overrepresentation at the 5′ and 3′ of Ori sites, respectively. Repeated GC-rich elements were detected, which are good predictors of Oris, suggesting that common sequence features are part of metazoan Oris. In the heterochromatic chromosome 4 of Drosophila, Oris correlated with HP1 binding sites. At the chromosome level, regions rich in Oris are early replicating, whereas Ori-poor regions are late replicating. The genome-wide analysis was coupled with a DNA combing analysis to unravel the organization of Oris. The results indicate that Oris are in a large excess, but their activation does not occur at random. They are organized in groups of site-specific but flexible origins that define replicons, where a single origin is activated in each replicon. This organization provides both site specificity and Ori firing flexibility in each replicon, allowing possible adaptation to environmental cues and cell fates