Centrosome centering and decentering by microtubule network rearrangement.

The centrosome is positioned at the cell center by pushing and pulling forces transmitted by microtubules (MTs). Centrosome decentering is often considered to result from asymmetric, cortical pulling forces exerted in particular by molecular motors on MTs and controlled by external cues affecting the cell cortex locally. Here we used numerical simulations to investigate the possibility that it could equally result from the redistribution of pushing forces due to a reorientation of MTs. We first showed that MT gliding along cell edges and pivoting around the centrosome regulate MT rearrangement and thereby direct the spatial distribution of pushing forces, whereas the number, dynamics, and stiffness of MTs determine the magnitude of these forces. By modulating these parameters, we identified different regimes, involving both pushing and pulling forces, characterized by robust centrosome centering, robust off-centering, or "reactive" positioning. In the last-named conditions, weak asymmetric cues can induce a misbalance of pushing and pulling forces, resulting in an abrupt transition from a centered to an off-centered position. Taken together, these results point to the central role played by the configuration of the MTs on the distribution of pushing forces that position the centrosome. We suggest that asymmetric external cues should not be seen as direct driver of centrosome decentering and cell polarization but instead as inducers of an effective reorganization of the MT network, fostering centrosome motion to the cell periphery

Geometrical and mechanical properties control actin filament organization.

The different actin structures governing eukaryotic cell shape and movement are not only determined by the properties of the actin filaments and associated proteins, but also by geometrical constraints. We recently demonstrated that limiting nucleation to specific regions was sufficient to obtain actin networks with different organization. To further investigate how spatially constrained actin nucleation determines the emergent actin organization, we performed detailed simulations of the actin filament system using Cytosim. We first calibrated the steric interaction between filaments, by matching, in simulations and experiments, the bundled actin organization observed with a rectangular bar of nucleating factor. We then studied the overall organization of actin filaments generated by more complex pattern geometries used experimentally. We found that the fraction of parallel versus antiparallel bundles is determined by the mechanical properties of actin filament or bundles and the efficiency of nucleation. Thus nucleation geometry, actin filaments local interactions, bundle rigidity, and nucleation efficiency are the key parameters controlling the emergent actin architecture. We finally simulated more complex nucleation patterns and performed the corresponding experiments to confirm the predictive capabilities of the model

A computational model of the early stages of acentriolar meiotic spindle assembly.

The mitotic spindle is an ensemble of microtubules responsible for the repartition of the chromosomal content between the two daughter cells during division. In metazoans, spindle assembly is a gradual process involving dynamic microtubules and recruitment of numerous associated proteins and motors. During mitosis, centrosomes organize and nucleate the majority of spindle microtubules. In contrast, oocytes lack canonical centrosomes but are still able to form bipolar spindles, starting from an initial ball that self-organizes in several hours. Interfering with early steps of meiotic spindle assembly can lead to erroneous chromosome segregation. Although not fully elucidated, this process is known to rely on antagonistic activities of plus end- and minus end-directed motors. We developed a model of early meiotic spindle assembly in mouse oocytes, including key factors such as microtubule dynamics and chromosome movement. We explored how the balance between plus end- and minus end-directed motors, as well as the influence of microtubule nucleation, impacts spindle morphology. In a refined model, we added spatial regulation of microtubule stability and minus-end clustering. We could reproduce the features of early stages of spindle assembly from 12 different experimental perturbations and predict eight additional perturbations. With its ability to characterize and predict chromosome individualization, this model can help deepen our understanding of spindle assembly

Geometrical and mechanical properties control actin filament organization.

The different actin structures governing eukaryotic cell shape and movement are not only determined by the properties of the actin filaments and associated proteins, but also by geometrical constraints. We recently demonstrated that limiting nucleation to specific regions was sufficient to obtain actin networks with different organization. To further investigate how spatially constrained actin nucleation determines the emergent actin organization, we performed detailed simulations of the actin filament system using Cytosim. We first calibrated the steric interaction between filaments, by matching, in simulations and experiments, the bundled actin organization observed with a rectangular bar of nucleating factor. We then studied the overall organization of actin filaments generated by more complex pattern geometries used experimentally. We found that the fraction of parallel versus antiparallel bundles is determined by the mechanical properties of actin filament or bundles and the efficiency of nucleation. Thus nucleation geometry, actin filaments local interactions, bundle rigidity, and nucleation efficiency are the key parameters controlling the emergent actin architecture. We finally simulated more complex nucleation patterns and performed the corresponding experiments to confirm the predictive capabilities of the model

DeXtrusion: automatic recognition of epithelial cell extrusion through machine learning in vivo

International audienceAccurately counting and localising cellular events from movies is an important bottleneck of high-content tissue/embryo live imaging. Here, we propose a new methodology based on deep learning that allows automatic detection of cellular events and their precise xyt localisation on live fluorescent imaging movies without segmentation. We focused on the detection of cell extrusion, the expulsion of dying cells from the epithelial layer, and devised DeXtrusion: a pipeline based on recurrent neural networks for automatic detection of cell extrusion/cell death events in large movies of epithelia marked with cell contour. The pipeline, initially trained on movies of the Drosophila pupal notum marked with fluorescent E-cadherin, is easily trainable, provides fast and accurate extrusion predictions in a large range of imaging conditions, and can also detect other cellular events, such as cell division or cell differentiation. It also performs well on other epithelial tissues with reasonable re-training. Our methodology could easily be applied for other cellular events detected by live fluorescent microscopy and could help to democratise the use of deep learning for automatic event detections in developing tissues

Dynamic reorganization of the actin cytoskeleton

Cellular processes, including morphogenesis, polarization, and motility, rely on a variety of actin-based structures. Although the biochemical composition and filament organization of these structures are different, they often emerge from a common origin. This is possible because the actin structures are highly dynamic. Indeed, they assemble, grow, and disassemble in a time scale of a second to a minute. Therefore, the reorganization of a given actin structure can promote the formation of another. Here, we discuss such transitions and illustrate them with computer simulations

Quantitative analysis of melanocyte migration in vitro based on automated cell tracking under phase contrast microscopy considering the combined influence of cell division and cell-matrix interactions.

International audienceThe aim of this study was to describe and analyze the regulation and spatio-temporal dynamics of melanocyte migration in vitro and its coupling to cell division and interaction with the matrix. The melanocyte lineage is particularly interesting because it is involved in both embryonic development and oncogenesis/metastasis (melanoma). Biological experiments were performed on two melanocyte cell lines established from wild-type and beta-catenin-transgenic mice (bcat*). The multi-functional beta-catenin molecule is known to be able to regulate the transcription of various genes involved in cell proliferation and migration, particularly in the melanocyte lineage. We also investigated fibronectin, an extra-cellular matrix protein that binds integrins, thereby providing adhesion points for cells and encouraging migration. As the migration of individual cells were followed, automated methods were required for processing the large amount of data generated by the time-lapse video-microscopy. A model-based approach for automated cell tracking was evaluated on a sample by comparison with manual tracking. This method was found reliable in studying overall cell behaviour. Its application to all the observed sequences provided insight into the factors affecting melanocyte migration in vitro: melanocytes of mutated form of beta-catenin showed higher division rates and no contact inhibition of growth was induced by the resulting increase in cell density. However, cell density limited the amplitude of cell displacements, although their motility was less affected. The high fibronectin concentration bound to substratum promoted cell migration and motility, the effect being more intense for wild-type cells than for cells with beta-catenin over-expression. During the division process, cell migration speed increased rapidly then decreased slowly. Analyses of such data is expected to lead both to biological answers and to a framework for a better modeling processes in the future

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

Acto-myosin network geometry defines centrosome position

International audienceThe centrosome is the main organizer of microtubules and as such, its position is a key determinant of polarized cell functions. As the name says, the default position of the centrosome is considered to be the cell geometrical center. However, the mechanism regulating centrosome positioning is still unclear and often confused with the mechanism regulating the position of the nucleus to which it is linked. Here, we used enucleated cells plated on adhesive micropatterns to impose regular and precise geometrical conditions to centrosome-microtubule networks. Although frequently observed there, the equilibrium position of the centrosome is not systematically at the cell geometrical center and can be close to cell edge. Centrosome positioning appears to respond accurately to the architecture and anisotropy of the actin network, which constitutes, rather than cell shape, the actual spatial boundary conditions the microtubule network is sensitive to. We found that the contraction of the actin network defines a peripheral margin in which microtubules appear bent by compressive forces. The progressive disassembly of the actin network at distance from the cell edges defines an inner zone where actin bundles were absent, where microtubules were more radially organized and where dynein concentration was higher. We further showed that the production of dynein-based forces on microtubules places the centrosome at the center of this zone. In conclusion, the spatial distribution of cell adhesion and the production of contractile forces define the architecture of the actin network with respect to which the centrosome-microtubule network is centered

Contribution of studied parameters on actin organization.

<p>(A) Schematic of a micropattern, and the corresponding <i>in vitro</i> actin organization observed with TIRF microscopy. Scale bar is 10 μm. (B) The contribution of the key elements of the model on the final organization of fibers in the simulation. (B1) Fibers that are nucleated locally but do not interact with each other fail to extend away from the micropattern, (B2) Adding the steric interaction with the parameters as calibrated in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004245#pcbi.1004245.g001" target="_blank">Fig 1</a> allows filaments to co-align and extend away from the nucleation zone. (B3) Setting the persistence length to 15 μm (cf. <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004245#pcbi.1004245.g002" target="_blank">Fig 2</a>) makes them extend further. (B4) Adding the nucleation effect (cf. <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004245#pcbi.1004245.g003" target="_blank">Fig 3</a>, H2) finally leads to realistic overall densities (final time is 250 s). (C) Predictions of the actin filament organization for novel nucleation patterns. Simulations (Top) were done on a set of 8 points, distant by 6 μm, and positioned on a square (left) or triangular (right) lattice. The 8-point units were repeated 9 times using periodic boundaries. (Bottom) Experimental verification of actin organization for the geometries used.</p