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Nedelec, F.

Publications and source records attributed to Nedelec, F..

16 recordsLinked to original sources
bioRxiv2023

The cell cycle controls spindle architecture in Arabidopsis by modulating the augmin pathway

To ensure an even segregation of chromosomes during somatic cell division, eukaryotes rely on specific microtubule structures called mitotic spindles. There are, however, striking differences in overall spindle organization among eukaryotic super groups, and in particular little is known about how spindle architecture is determined in plants. As a foundation for our work, we have measured prime characteristics of Arabidopsis mitotic spindles and built a three-dimensional dynamic model of the Arabidopsis mitotic spindle using Cytosim. Next, we identified the cell-cycle regulator CYCLIN-DEPENDENT KINASE B1 (CDKB1) together with its cyclin partner CYCB3;1 as key regulators of spindle shape and organization in Arabidopsis. Loss of CDKB1 function resulted in a high number of astral microtubules that are normally absent from plant spindles, as opposed to animal ones. We identified an augmin complex member, ENDOSPERM DEFECTIVE1 (EDE1), as a substrate of the CDKB1;1-CYCB3;1 complex. A non-phosphorylatable mutant of EDE1 displayed spindles with extended pole-to-pole distance, resembling the phenotypes of cycb3;1 and cdkb1 mutants. Moreover, we found that the mutated EDE1 version associated less efficiently with spindle microtubules. Consistently, reducing the level of augmin in Cytosim simulations largely recapitulated the spindle phenotypes observed in cycb3;1 and cdkb1 mutants. Our results emphasize the importance of cell cycle-dependent phospho-control of the mitotic spindle in plant cells. They also support the validity of our computational model as a framework for the exploration of mechanisms controlling the organization of the spindle in plants and in other species.

developmental biology↗
bioRxiv2022

Effects of microtubule length and crowding on active microtubule network organization

Active filament networks can organize into various dynamic architectures driven by crosslinking motors. Densities and kinetic properties of motors and microtubules have been shown previously to determine active microtubule network self-organization, but the effects of other control parameters are less understood. Using computer simulations, we study here how microtubule lengths and crowding effects determine active network architecture and dynamics. We find that attractive interaction mimicking crowding effects or long microtubules both promote the formation of nematic networks of extensile bundles instead of contractile networks. When microtubules are very long and the network is highly percolated, a new isotropically motile network state resembling a crawling mesh is predicted. Using in vitro reconstitutions, we confirm the existence of this crawling mesh experimentally. These results provide a better understanding of how active microtubule network organization can be controlled, with implications for cell biology and active materials in general.

biophysics↗
bioRxiv2022

Contractile ring composition dictates kinetics of in silico contractility

Constriction kinetics of the cytokinetic ring are expected to depend on dynamic adjustment of ring composition, but the impact of component abundance dynamics on ring constriction is understudied. Computational models generally assume that contractile networks maintain constant composition. To test how compositional dynamics affect constriction kinetics, we first measured F-actin, non-muscle myosin II, septin, and anillin during C. elegans zygotic mitosis. A custom microfluidic device that positioned the cell with the division plane parallel to a light sheet allowed even illumination of the cytokinetic ring. Measured component abundances were implemented in an agent-based 3D model of a membrane-associated contractile ring. With constant network composition, constriction occurred with biologically unrealistic kinetics. Measured changes in component quantities elicited realistic constriction kinetics. Simulated networks were more sensitive to changes in motor and filament amounts, than that of crosslinkers and tethers. Our findings highlight the importance of network composition for actomyosin contraction kinetics. SummaryWe created a microfluidic device compatible with high numerical aperture light sheet microscopy to measure cytokinetic ring component abundance in the C. elegans zygote. Implementing measured dynamics into our three-dimensional agent-based model of a contractile ring elicited biologically realistic kinetics.

cell biology↗
bioRxiv2022

Crosslinker design determines microtubule network organization by opposing motors

During cell division, crosslinking motors determine the architecture of the spindle, a dynamic microtubule network that segregates the chromosomes. It is unclear how motors with opposite directionality coordinate to drive both contractile and extensile behaviors in the spindle. Particularly, the impact of different crosslinker designs on network self-organization is not understood, limiting our understanding of self-organizing structures in cells, but also our ability to engineer new active materials. Here, we use experiment and theory to examine active microtubule networks driven by mixtures of motors with opposite directionality and different crosslinker design. We find that although the kinesin-14 HSET causes network contraction when dominant, it can also assist the opposing kinesin-5 KIF11 to generate extensile networks. This bifunctionality results from HSETs asymmetric design, distinct from symmetric KIF11. These findings expand the set of rules underlying patterning of active microtubule assemblies and allow a better understanding of motor cooperation in the spindle. SIGNIFICANCE STATEMENTDuring cell division, the spindle apparatus segregates duplicated chromosomes for their inheritance by the daughter cells. The spindle is a highly interconnected network of microtubule filaments that are crosslinked by different types of molecular motors. How the different motors cooperate to organize the spindle network is not understood. Here, we show that an asymmetric crosslinker design can confer bifunctionality to a mitotic motor in the presence of other motors. The asymmetric motor supports both extensile and contractile microtubule network behaviors as observed in different parts of the spindle. These findings define new rules controlling the generation of active microtubule networks and allow us to better understand how motors cooperate to organize the correct spindle architecture when a cell divides.

biophysics↗
bioRxiv2021

Microtubule rescue at midzone edges promotes overlap stability and prevents spindle collapse during anaphase B

During anaphase B, molecular motors slide interpolar microtubules to elongate the mitotic spindle, contributing to the separation of chromosomes. However, sliding of antiparallel microtubules reduces their overlap, which may lead to spindle breakage, unless microtubules grow to compensate sliding. How sliding and growth are coordinated is still poorly understood. In this study, we have used the fission yeast S. pombe to measure microtubule dynamics during anaphase B. We report that the coordination of microtubule growth and sliding relies on promoting rescues at the midzone edges. This makes microtubules stable from pole to midzone, while their distal parts including the plus ends alternate between assembly and disassembly. Consequently, the midzone keeps a constant length throughout anaphase, enabling sustained sliding without the need for a precise regulation of microtubule growth speed. Additionally, we found that in S. pombe, which undergoes closed mitosis, microtubule growth speed decreases when the nuclear membrane wraps around the spindle midzone.

cell biology↗
bioRxiv2019

Theory of antiparallel microtubule overlap stabilization by motors and diffusible crosslinkers

Antiparallel microtubule bundles are essential structural elements of many cytoskeletal structures, for instance the mitotic spindle. In such bundles, neighbouring microtubules are bonded by specialised crosslinkers of the Ase1/PRC1/MAP65 family that can diffuse longitudinally along microtubules. Similarly, some kinesin motors implicated in bundle formation have a diffusible tail allowing them to slide passively along microtubules. We develop here a theory of two microtubules connected by motors and diffusible connectors, in different configurations that can be realized experimentally. In all cases, the microtubule sliding speed derived analytically is validated by stochastic simulations and used to discuss recent experimental results, such as force generation by kinesin-14, and overlap stabilization by Ase1. Some systems can produce steady overlaps that are determined by the density of crosslinkers on the microtubule lattice. This property naturally leads to robust coordination between sliding and growth in dynamic bundles of microtubules, an essential property in mitosis.

cell biology↗
bioRxiv2019

Bond type and discretization of non-muscle myosin II are critical for simulated contractile dynamics

Molecular motors drive cytoskeletal rearrangements to change cell shape. Myosins are the motors that move, crosslink, and modify the actin cytoskeleton. The primary force generator in contractile actomyosin networks is non-muscle myosin II (NMMII), a molecular motor that assembles into ensembles that bind, slide, and crosslink actin filaments (F-actin). The multivalence of NMMII ensembles and their multiple roles have confounded the resolution of crucial questions including how the number of NMMII subunits affects dynamics, and what affects the relative contribution of ensembles crosslinking versus motoring activities. Since biophysical measurements of ensembles are sparse, modeling of actomyosin networks has aided in discovering the complex behaviors of NMMII ensembles. Myosin ensembles have been modeled via several strategies with variable discretization/coarse-graining and unbinding dynamics, and while general assumptions that simplify motor ensembles result in global contractile behaviors, it remains unclear which strategies most accurately depict cellular activity. Here, we used an agent-based platform, Cytosim, to implement several models of NMMII ensembles. Comparing the effects of bond type, we found that ensembles of catch-slip and catch motors were the best force generators and binders of filaments. Slip motor ensembles were capable of generating force but unbound frequently, resulting in slower contractile rates of contractile networks. Coarse-graining of these ensemble types from two sets of 16 motors on opposite ends of a stiff rod to two binders, each representing 16 motors, reduced force generation, contractility, and the total connectivity of filament networks for all ensemble types. A parallel cluster model (PCM) previously used to describe ensemble dynamics via statistical mechanics, allowed better contractility with coarse-graining, though connectivity was still markedly reduced for this ensemble type with coarse-graining. Together our results reveal substantial trade-offs associated with the process of coarse-graining NMMII ensembles and highlight the robustness of discretized catch-slip ensembles in modeling actomyosin networks. STATEMENT OF SIGNIFICANCEAgent-based simulations of contractile networks allow us to explore the mechanics of actomyosin contractility, which drives many cell shape changes including cytokinesis, the final step of cell division. Such simulations should be able to predict the mechanics and dynamics of non-muscle contractility, however recent work has highlighted a lack of consensus on how to best model the non-muscle myosin II. These ensembles of approximately 32 motors are the key components responsible for driving contractility. Here, we explored different methods for modeling non-muscle myosin II ensembles within the context of contractile actomyosin networks. We show that the level of coarse-graining and the choice of unbinding model used to model motor unbinding under load indeed has profound effects on contractile network dynamics.

cell biology↗
bioRxiv2018

Cytoskeleton mechanics determine resting size and activation dynamics of platelets.

Platelets are cell fragments of various size that help maintain hemostasis. The way platelets respond during a clotting process is known to depend on their size, with important physiological consequences. We characterized the cytoskeleton of platelets as a function of their size. In resting Human and Mice platelets, we find a quadradic law between the size of a platelet and the amount of microtubule polymer it contains. We further estimate the length and number of microtubules in the marginal band using Electron and Super-resolution microscopy. In platelets activated with ADP, the marginal band coils as a consequence of cortical contraction driven by actin. We observe that this elastic coiling response is accompanied by a reversible shortening of the marginal band. Moreover, larger platelets have a higher propensity to coil. These results establish the dynamic equilibrium that is responsible for platelet size and differential response on a more quantitative level.\n\nHighlightsO_LIPlatelet size scales consistently with amount of polymerized tubulin in both mouse and human.\nC_LIO_LIPolymerized actin is required for ADP-induced marginal band coiling.\nC_LIO_LIUpon activation, the marginal band exhibits a reversible visco-elastic response involving shortening.\nC_LIO_LILarger marginal bands have a higher propensity to coil than shorter ones.\nC_LI\n\nIn briefThe cytoskeleton is adapted to platelet size and its mechanical properties determine propensity of a platelet to undergo morphological changes in response to agonists.

cell biology↗
bioRxiv2018

Polarity sorting drives remodeling of actin-myosin networks

Cytoskeletal networks of actin filaments and myosin motors drive many dynamic cell processes. A key characteristic of these networks is their contractility. Despite intense experimental and theoretical efforts, it is not clear what mechanism favors network contraction over expansion. Recent work points to a dominant role for the nonlinear mechanical response of actin filaments, which can withstand stretching but buckle upon compression. Here we present an alternative mechanism. We study how interactions between actin and myosin-2 at the single filament level translate into contraction at the network scale by performing time-lapse imaging on reconstituted quasi-2D-networks mimicking the cell cortex. We observe myosin end-dwelling after it runs processively along actin filaments. This leads to transport and clustering of actin filament ends and the formation of transiently stable bipolar structures. Further we show that myosin-driven polarity sorting leads to polar actin aster formation, which act as contractile nodes that drive contraction in crosslinked networks. Computer simulations comparing the roles of the end-dwelling mechanism and a buckling-dependent mechanism show that the relative contribution of end-dwelling contraction increases as the network mesh-size decreases.

biophysics↗
bioRxiv2018

F-actin patches nucleated on chromosomes coordinate capture by microtubules in oocyte meiosis

Capture of each and every chromosome by spindle microtubules is essential to prevent chromosome loss and aneuploidy. In somatic cells, astral microtubules search and capture chromosomes forming lateral attachments to kinetochores. However, this mechanism alone is insufficient in large oocytes. We have previously shown that a contractile F-actin network is additionally required to collect chromosomes scattered in the 70-m starfish oocyte nucleus. How this F-actin-driven mechanism is coordinated with microtubule capture remained unknown. Here, we show that after nuclear envelope breakdown Arp2/3-nucleated F-actin patches form around chromosomes in a Ran-GTP-dependent manner, and we propose that these structures sterically block kinetochore-microtubule attachments. Once F-actin-driven chromosome transport is complete, coordinated disassembly of these F-actin patches allows synchronous capture by microtubules. Our observations indicate that this coordination is necessary, as early capture of chromosomes by microtubules would interfere with F-actin-driven transport leading to chromosome loss and formation of aneuploid eggs.

cell biology↗
bioRxiv2017

Systematic analysis of the molecular architecture of endocytosis reveals a nanoscale actin nucleation template that drives efficient vesicle formation

Clathrin-mediated endocytosis is an essential cellular function in all eukaryotes that is driven by a self-assembled macromolecular machine of over 50 different proteins in tens to hundreds of copies. How these proteins are organized to produce endocytic vesicles with high precision and efficiency is not understood. Here, we developed high-throughput superresolution microscopy to reconstruct the nanoscale structural organization of 23 endocytic proteins from over 100,000 endocytic sites in yeast. We found that proteins assemble by radially-ordered recruitment according to function. WASP family proteins form a circular nano-scale template on the membrane to spatially control actin nucleation during vesicle formation. Mathematical modeling of actin polymerization showed that this WASP nano-template creates sufficient force for membrane invagination and substantially increases the efficiency of endocytosis. Such nanoscale pre-patterning of actin nucleation may represent a general design principle for directional force generation in membrane remodeling processes such as during cell migration and division.

cell biology↗
bioRxiv2017

Crosslinkers both drive and brake cytoskeletal remodeling and furrowing in cytokinesis

Cytokinesis and other cell shape changes are driven by the actomyosin contractile cytoskeleton. The molecular rearrangements that bring about contractility in non-muscle cells are currently debated. Specifically, both filament sliding by myosin motors, as well as cytoskeletal crosslinking by myosins and non-motor crosslinkers, are thought to promote contractility. Here, we examined how the abundance of motor and non-motor crosslinkers controls the speed of cytokinetic furrowing. We built a minimal model to simulate the contractile dynamics of the C. elegans zygote cytokinetic ring. This model predicted that intermediate levels of non-motor crosslinkers would allow maximal contraction speed, which we found to be the case for the scaffold protein anillin, in vivo. Our model also demonstrated a non-linear relationship between the abundance of motor ensembles and contraction speed. In vivo, thorough depletion of non-muscle myosin II delayed furrow initiation, slowed F-actin alignment, and reduced maximum contraction speed, but partial depletion allowed faster-than-expected kinetics. Thus, both motor and non-motor crosslinkers promote cytokinetic ring closure when present at low levels, but act as a brake when present at higher levels. Together, our findings extend the growing appreciation for the roles of crosslinkers, but reveal that they not only drive but also brake cytoskeletal remodeling.

cell biology↗
bioRxiv2017

A Theory That Predicts Behaviors Of Disordered Cytoskeletal Networks

Morphogenesis in animal tissues is largely driven by tensions of actomyosin networks, generated by an active contractile process that can be reconstituted in vitro. Although the network components and their properties are known, the requirements for contractility are still poorly understood. Here, we describe a theory that predicts whether an isotropic network will contract, expand, or conserve its dimensions. This analytical theory correctly predicts the behavior of simulated networks consisting of filaments with varying combinations of connectors, and reveals conditions under which networks of rigid filaments are either contractile or expansile. Our results suggest that pulsatility is an intrinsic behavior of contractile networks if the filaments are not stable but turn over. The theory offers a unifying framework to think about mechanisms of contractions or expansion. It provides a foundation for the study of a broad range of processes involving cytoskeletal networks, and a basis for designing synthetic networks.

cell biology↗
bioRxiv2016

Mechanism of Nuclear Movements in a Multinucleated Cell

Multinucleated cells are important in many organisms but the mechanisms governing the movements of nuclei sharing a common cytoplasm are not understood. In the hyphae of the plant pathogenic fungus Ashbya gossypii, nuclei move back and forth, occasionally bypassing each other, and, preventing the formation of nuclear clusters, this is essential for genetic stability. These movements depend on cytoplasmic microtubules emanating from the nuclei, that are pulled by dynein motors anchored at the cortex. Using 3D stochastic simulations with parameters constrained by the literature, we predict the cortical anchors density from the characteristics of nuclear movements. Altogether, the model accounts for the complex nuclear movements seen in vivo, using a minimal set of experimentally determined ingredients. Interestingly, these ingredients power the oscillations of the anaphase spindle in budding yeast, but in A. gossypii this system is not restricted to a specific nuclear cycle stage, possibly as a result of adaptation to hyphal growth and multinuclearity.

cell biology↗
bioRxiv2016

ConfocalGN : a minimalistic confocal image simulator.

We developed a user-friendly software to generate synthetic confocal microscopy images from a ground truth specified as a 3D bitmap with pixels of arbitrary size. The software can analyze a real confocal stack to derivate noise parameters and will use them directly to generate new images with similar noise characteristics. Such synthetic images can then be used to assert the quality and robustness of an image analysis pipeline, as well as be used to train machine-learning image analysis procedures. We illustrate the approach with closed curves corresponding to the microtubule ring present in blood platelets.\n\nAvailability and implementationConfocalGN is written in MATLAB but does not require any toolbox. The source code is distributed under the GPL 3.0 licence on https://github.com/SergeDmi/ConfocalGN.

bioinformatics↗
bioRxiv2016

Balance of microtubule stiffness and cortical tension determines the size of blood cells with marginal band across species

The fast blood stream of animals is associated with large shear stresses. Consequently, blood cells have evolved a special morphology and a specific internal architecture allowing them to maintain their integrity over several weeks. For instance, non-mammalian red blood cells, mammalian erythroblasts and platelets have a peripheral ring of microtubules, called the marginal band, that flattens the overall cell morphology by pushing on the cell cortex. In this article, we model how the shape of these cells stems from the balance between marginal band elasticity and cortical tension. We predict that the diameter of the cell scales with the total microtubule polymer, and verify the predicted law across a wide range of species. Our analysis also shows that the combination of the marginal band rigidity and cortical tension increases the ability of the cell to withstand forces without deformation. Finally, we model the marginal band coiling that occurs during the disc-to-sphere transition observed for instance at the onset of blood platelet activation. We show that when cortical tension increases faster than crosslinkers can unbind, the marginal band will coil, whereas if the tension increases slower, the marginal band may shorten as microtubules slide relative to each other.\n\nSignificance StatementMany blood cells have a discoidal shape, which is essential for them to function properly within the organism. For some cells, such as blood platelets, this shape is due to the interplay between the elasticity of the marginal band, which is a closed ring of stiff filaments called microtubules, and the tension of the cell cortex, a polymer scaffold associated with the plasma membrane. Dmitrieff et al. examined how cell size is determined by the mechanical balance between these two components. Remarkably, the theory is confirmed over nearly three orders of magnitudes, by data collected from 25 species. The theory also shows how the composite structure is adapted to resist transient mechanical challenges, as encountered in the blood stream.

biophysics↗