Author: "Laurent Blanchoin" - Searchworks@Jio Institute Digital Library Search Results

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188 results on '"Laurent Blanchoin"'

1. Structural and biochemical evidence for the emergence of a calcium-regulated actin cytoskeleton prior to eukaryogenesis

Author: Caner Akıl, Linh T. Tran, Magali Orhant-Prioux, Yohendran Baskaran, Yosuke Senju, Shuichi Takeda, Phatcharin Chotchuang, Duangkamon Muengsaen, Albert Schulte, Edward Manser, Laurent Blanchoin, and Robert C. Robinson
Subjects: Biology (General), QH301-705.5
Abstract: Calcium-regulated actin filament assembly predates eukaryogenesis and was present in the last common ancestor of Asgard archaea Loki, Heim, and Thorarchaeota.
Published: 2022
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2. Kinesin-6 Klp9 orchestrates spindle elongation by regulating microtubule sliding and growth

Author: Lara Katharina Krüger, Matthieu Gélin, Liang Ji, Carlos Kikuti, Anne Houdusse, Manuel Théry, Laurent Blanchoin, and Phong T Tran
Subjects: mitosis, mitotic spindle, Kinesin-6, microtubule polymerisation, microtubule sliding, XMAP215, Medicine, Science, Biology (General), QH301-705.5
Abstract: Mitotic spindle function depends on the precise regulation of microtubule dynamics and microtubule sliding. Throughout mitosis, both processes have to be orchestrated to establish and maintain spindle stability. We show that during anaphase B spindle elongation in Schizosaccharomyces pombe, the sliding motor Klp9 (kinesin-6) also promotes microtubule growth in vivo. In vitro, Klp9 can enhance and dampen microtubule growth, depending on the tubulin concentration. This indicates that the motor is able to promote and block tubulin subunit incorporation into the microtubule lattice in order to set a well-defined microtubule growth velocity. Moreover, Klp9 recruitment to spindle microtubules is dependent on its dephosphorylation mediated by XMAP215/Dis1, a microtubule polymerase, creating a link between the regulation of spindle length and spindle elongation velocity. Collectively, we unravel the mechanism of anaphase B, from Klp9 recruitment to the motors dual-function in regulating microtubule sliding and microtubule growth, allowing an inherent coordination of both processes.
Published: 2021
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3. Loss of Ena/VASP interferes with lamellipodium architecture, motility and integrin-dependent adhesion

Author: Julia Damiano-Guercio, Laëtitia Kurzawa, Jan Mueller, Georgi Dimchev, Matthias Schaks, Maria Nemethova, Thomas Pokrant, Stefan Brühmann, Joern Linkner, Laurent Blanchoin, Michael Sixt, Klemens Rottner, and Jan Faix
Subjects: cell migration, Ena/VASP proteins, lamellipodia, microspikes, filopodia, cell adhesion, Medicine, Science, Biology (General), QH301-705.5
Abstract: Cell migration entails networks and bundles of actin filaments termed lamellipodia and microspikes or filopodia, respectively, as well as focal adhesions, all of which recruit Ena/VASP family members hitherto thought to antagonize efficient cell motility. However, we find these proteins to act as positive regulators of migration in different murine cell lines. CRISPR/Cas9-mediated loss of Ena/VASP proteins reduced lamellipodial actin assembly and perturbed lamellipodial architecture, as evidenced by changed network geometry as well as reduction of filament length and number that was accompanied by abnormal Arp2/3 complex and heterodimeric capping protein accumulation. Loss of Ena/VASP function also abolished the formation of microspikes normally embedded in lamellipodia, but not of filopodia capable of emanating without lamellipodia. Ena/VASP-deficiency also impaired integrin-mediated adhesion accompanied by reduced traction forces exerted through these structures. Our data thus uncover novel Ena/VASP functions of these actin polymerases that are fully consistent with their promotion of cell migration.
Published: 2020
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4. A key function for microtubule-associated-protein 6 in activity-dependent stabilisation of actin filaments in dendritic spines

Author: Leticia Peris, Mariano Bisbal, José Martinez-Hernandez, Yasmina Saoudi, Julie Jonckheere, Marta Rolland, Muriel Sebastien, Jacques Brocard, Eric Denarier, Christophe Bosc, Christophe Guerin, Sylvie Gory-Fauré, Jean Christophe Deloulme, Fabien Lanté, Isabelle Arnal, Alain Buisson, Yves Goldberg, Laurent Blanchoin, Christian Delphin, and Annie Andrieux
Subjects: Science
Abstract: Microtubule-associated protein 6 (MAP6) is known to be important for synaptic plasticity and cognition, supposedly via interaction with microtubules. Here, the authors found that MAP6 is crucial for the stabilisation of enlarged synapses through its association with a different cytoskeletal element, actin.
Published: 2018
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5. Network heterogeneity regulates steering in actin-based motility

Author: Rajaa Boujemaa-Paterski, Cristian Suarez, Tobias Klar, Jie Zhu, Christophe Guérin, Alex Mogilner, Manuel Théry, and Laurent Blanchoin
Subjects: Science
Abstract: Protrusive cellular structures contain a heterogeneous density of actin, but whether this influences motility is not known. Using an in vitro system and modelling, here the authors show that local actin monomer depletion and network architecture can tune the rate of network growth to impose steering during motility.
Published: 2017
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6. Quantitative regulation of the dynamic steady state of actin networks

Author: Angelika Manhart, Téa Aleksandra Icheva, Christophe Guerin, Tobbias Klar, Rajaa Boujemaa-Paterski, Manuel Thery, Laurent Blanchoin, and Alex Mogilner
Subjects: actin, ADF/Cofilin, actin network length, actin disassembly, Medicine, Science, Biology (General), QH301-705.5
Abstract: Principles of regulation of actin network dimensions are fundamentally important for cell functions, yet remain unclear. Using both in vitro and in silico approaches, we studied the effect of key parameters, such as actin density, ADF/Cofilin concentration and network width on the network length. In the presence of ADF/Cofilin, networks reached equilibrium and became treadmilling. At the trailing edge, the network disintegrated into large fragments. A mathematical model predicts the network length as a function of width, actin and ADF/Cofilin concentrations. Local depletion of ADF/Cofilin by binding to actin is significant, leading to wider networks growing longer. A single rate of breaking network nodes, proportional to ADF/Cofilin density and inversely proportional to the square of the actin density, can account for the disassembly dynamics. Selective disassembly of heterogeneous networks by ADF/Cofilin controls steering during motility. Our results establish general principles on how the dynamic steady state of actin network emerges from biochemical and structural feedbacks.
Published: 2019
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7. Actin nucleation at the centrosome controls lymphocyte polarity

Author: Dorian Obino, Francesca Farina, Odile Malbec, Pablo J. Sáez, Mathieu Maurin, Jérémie Gaillard, Florent Dingli, Damarys Loew, Alexis Gautreau, Maria-Isabel Yuseff, Laurent Blanchoin, Manuel Théry, and Ana-Maria Lennon-Duménil
Subjects: Science
Abstract: Cell polarity is marked by re-orientation of the centrosome, but the mechanisms governing centrosome polarization are poorly understood. Here Obino et al. show that in lymphocytes centrosome-associated Arp2/3 nucleates actin that tethers the centrosome to the nucleus; activation depletes Arp2/3 from the centrosome and frees it from the nucleus.
Published: 2016
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8. Dynamic reorganization of the actin cytoskeleton [version 1; referees: 2 approved]

Author: Gaëlle Letort, Hajer Ennomani, Laurène Gressin, Manuel Théry, and Laurent Blanchoin
Subjects: Cell Adhesion, Cell Signaling, Cytoskeleton, Macromolecular Chemistry, Protein Chemistry & Proteomics, Medicine, Science
Abstract: 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.
Published: 2015
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9. Geometrical and mechanical properties control actin filament organization.

Author: Gaëlle Letort, Antonio Z Politi, Hajer Ennomani, Manuel Théry, Francois Nedelec, and Laurent Blanchoin
Subjects: Biology (General), QH301-705.5
Abstract: 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.
Published: 2015
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10. Microtubules under mechanical pressure can breach dense actin networks

Author: Matthieu Gélin, Alexandre Schaeffer, Jérémie Gaillard, Christophe Guérin, Benoit Vianay, Magali Orhant-Prioux, Marcus Braun, Christophe Leterrier, Laurent Blanchoin, and Manuel Théry
Abstract: The crosstalk between actin network and microtubules is key to the establishment of cell polarity. It ensures that the asymmetry of actin architec ture along cell periphery directs the organization of microtubules in cell interior. In particular, the way the two networks are physically inter-twined regulates the spatial organization and the distribution of forces in the microtubule network. While their biochemical crosstalk is getting uncovered, their mechanical crosstalk is still poorly understood. Here we designed an in vitro reconstitution assay to study the physical interaction between dynamic microtubules with various structures made of actin fil aments. We found that microtubules can align and move by their polymerization force along linear bundles of actin filaments. But they cannot enter dense and branched actin meshworks, such as those found in the lamellipodium along cell periphery. However, when microtubules are immobilized, by their crosslinking with actin structures or others means, the force of polymerization builds up pressure in the microtubules that is sufficient to allow them to breach and penetrate these dense actin meshworks. This mechanism may explain the final progression of microtubules up to cell periphery through the denser parts of the actin network.
Published: 2023
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11. Microtubules self-repair in living cells

Author: Morgan Gazzola, Alexandre Schaeffer, Ciarán Butler-Hallissey, Karoline Friedl, Benoit Vianay, Jérémie Gaillard, Christophe Leterrier, Laurent Blanchoin, Manuel Théry, Ecotaxie, microenvironnement et développement lymphocytaire (EMily (UMR_S_1160 / U1160)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Institut de Recherche Saint-Louis - Hématologie Immunologie Oncologie (Département de recherche de l’UFR de médecine, ex- Institut Universitaire Hématologie-IUH) (IRSL), Université Paris Cité (UPCité), CytoMorphoLab, Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Hopital Saint-Louis [AP-HP] (AP-HP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Institut de neurophysiopathologie (INP), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Abbelight, Bettencourt-Schueller Foundation, Emergence program of the Ville de Paris, Schlumberger Foundation for Education and Research, ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), European Project: 771599,ICEBERG, European Project: 741773,AAA, Martin-Laffon, Jacqueline, CBH-EUR-GS - - CBH-EUR-GS2017 - ANR-17-EURE-0003 - EURE - VALID, Grenoble Alliance for Integrated Structural Cell Biology - - GRAL2010 - ANR-10-LABX-0049 - LABX - VALID, Exploration below the tip of the microtubule - ICEBERG - 771599 - INCOMING, and Adaptive Actin Architectures - AAA - 741773 - INCOMING
Subjects: renewal, remnants, self-repair, shaft, MESH: Guanosine Triphosphate, MESH: Microtubules, MESH: Cytoplasm, GTP islands, MESH: Tubulin, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, General Biochemistry, Genetics and Molecular Biology, MESH: Polymers, tubulin, rescue, MESH: Actin Cytoskeleton, General Agricultural and Biological Sciences, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, lattice, microtubule
Abstract: International audience; Microtubule self-repair has been studied both in vitro and in vivo as an underlying mechanism of microtubule stability. The turnover of tubulin dimers along the microtubule has challenged the pre-existing dogma that only growing ends are dynamic. However, although there is clear evidence of tubulin incorporation into the shaft of polymerized microtubules in vitro, the possibility of such events occurring in living cells remains uncertain. In this study, we investigated this possibility by microinjecting purified tubulin dimers labeled with a red fluorophore into the cytoplasm of cells expressing GFP-tubulin. We observed the appearance of red dots along the pre-existing green microtubule within minutes. We found that the fluorescence intensities of these red dots were inversely correlated with the green signal, suggesting that the red dimers were incorporated into the microtubules and replaced the pre-existing green dimers. Lateral distance from the microtubule center was similar to that in incorporation sites and in growing ends. The saturation of the size and spatial frequency of incorporations as a function of injected tubulin concentration and post-injection delay suggested that the injected dimers incorporated into a finite number of damaged sites. By our low estimate, within a few minutes of the injections, free dimers incorporated into major repair sites every 70 μm of microtubules. Finally, we mapped the location of these sites in micropatterned cells and found that they were more concentrated in regions where the actin filament network was less dense and where microtubules exhibited greater lateral fluctuations.
Published: 2023
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12. Friction patterns guide actin network contraction

Author: Alexandra Colin, Magali Orhant-Prioux, Christophe Guérin, Mariya Savinov, Ilaria Scarfone, Aurelien Roux, Enrique M. De La Cruz, Alex Mogilner, Manuel Théry, and Laurent Blanchoin
Abstract: The shape of cells is the outcome of the balance of inner forces produced by the actomyosin network and the resistive forces produced by cell adhesion to their environment. The specific contributions of contractile, anchoring and friction forces to network deformation rate and orientation are difficult to disentangle in living cells where they influence each other. Here, we reconstituted contractile acto-myosin networksin vitroto study specifically the role of the friction forces between the network and its anchoring substrate. To modulate the magnitude and spatial distribution of friction forces, we micropatterned actin nucleation promoting factors on glass or on a lipid bilayer. We adapted their concentrations on each surface to induce the assembly of actin networks of similar densities, and compare the deformation of the network toward the centroid of the pattern shape upon myosin-induced contraction. We found that actin network deformation was faster and more coordinated on lipid bilayers than on glass, showing the resistance of friction to network contraction. To further study the role of the spatial distribution of these friction forces, we designed heterogeneous micropatterns made of glass and lipids. The deformation upon contraction was no longer symmetric but biased toward the region of higher friction. Furthermore, we showed that the pattern of friction could robustly drive network contraction and dominate the contribution of asymmetric distributions of myosins. Therefore, we demonstrate that during contraction both the active and resistive forces are essential to direct the actin network deformation.Significance statementCell shape changes are controlled by complex sets of mechanical forces of various origins. Numerous studies have been dedicated to the role of active forces, originating from molecular motors and filament polymerization, but much less is known about the guiding role of resistive forces. Here we show that a non-uniform distribution of friction forces between a contracting acto-myosin network and its underlying substrate can direct its deformation as it contracts. Our results suggest that the contribution of resistive forces, such as anchoring forces but also less specific viscous forces along cell surface, can be as significant as those of active forces in driving network deformation and should be considered in mechanical models describing the regulation of cell shape and movements.
Published: 2022
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13. Actin Architecture Steers Microtubules in Active Cytoskeletal Composite

Author: Ondřej Kučera, Jérémie Gaillard, Christophe Guérin, Clothilde Utzschneider, Manuel Théry, Laurent Blanchoin, CytoMorphoLab, Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), European Project: 771599,ICEBERG, and European Project: 741773,AAA
Subjects: cytoskeletal composite, MESH: Microtubules, Mechanical Engineering, Bioengineering, General Chemistry, network architecture, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Condensed Matter Physics, MESH: Actins, Microtubules, Actins, gliding assay, Actin Cytoskeleton, actin filaments, MESH: Cytoskeleton, Nanotechnology, General Materials Science, MESH: Actin Cytoskeleton, MESH: Nanotechnology, Cytoskeleton
Abstract: International audience; Motility assays use surface-immobilized molecular motors to propel cytoskeletal filaments. They have been widely used to characterize motor properties and their impact on cytoskeletal self-organization. Moreover, the motility assays are a promising class of bioinspired active tools for nanotechnological applications. While these assays involve controlling the filament direction and speed, either as a sensory readout or a functional feature, designing a subtle control embedded in the assay is an ongoing challenge. Here, we investigate the interaction between gliding microtubules and networks of actin filaments. We demonstrate that the microtubule's behavior depends on the actin architecture. Both unbranched and branched actin decelerate microtubule gliding; however, an unbranched actin network provides additional guidance and effectively steers the microtubules. This effect, which resembles the recognition of cortical actin by microtubules, is a conceptually new means of controlling the filament gliding with potential application in the design of active materials and cytoskeletal nanodevices.
Published: 2022
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14. Actin–microtubule dynamic composite forms responsive active matter with memory

Author: Ondřej Kučera, Jérémie Gaillard, Christophe Guérin, Manuel Théry, Laurent Blanchoin, CytoMorphoLab, Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Commissariat à L'Energie Atomique Et Aux Energies Alternatives (CEA), Direction de La Recherche Fondamentale (DRF), Service de Recherche en Hémato-Immunologie (SRHI), Hôpital Saint-Louis, ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), European Project: 771599,ICEBERG, and European Project: 741773,AAA
Subjects: Multidisciplinary, Protein Stability, MESH: Microtubules, cytoskeleton, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, MESH: Actins, Microtubules, active materials, Actins, Actin Cytoskeleton, MESH: Protein Stability, MESH: Cytoskeleton, MESH: Actin Cytoskeleton, structural memory
Abstract: Active cytoskeletal materials in vitro demonstrate self-organising properties similar to those observed in their counterparts in cells. However, the search to emulate phenomena observed in the living matter has fallen short of producing a cytoskeletal network that would be structurally stable yet possessing adaptive plasticity. Here, we address this challenge by combining cytoskeletal polymers in a composite, where self-assembling microtubules and actin filaments collectively self-organise due to the activity of microtubules-percolating molecular motors. We demonstrate that microtubules spatially organise actin filaments that in turn guide microtubules. The two networks align in an ordered fashion using this feedback loop. In this composite, actin filaments can act as structural memory and, depending on the concentration of the components, microtubules either write this memory or get guided by it. The system is sensitive to external stimuli suggesting possible autoregulatory behaviour in changing mechanochemical environment. We thus establish artificial active actin-microtubule composite as a system demonstrating architectural stability and plasticity.
Published: 2022
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15. Actin network architecture can ensure robust centering or sensitive decentering of the centrosome

Author: Shohei Yamamoto, Jérémie Gaillard, Benoit Vianay, Christophe Guerin, Magali Orhant‐Prioux, Laurent Blanchoin, Manuel Théry, CytoMorphoLab, Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Immunologie humaine, physiopathologie & immunothérapie (HIPI (UMR_S_976 / U976)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Fellowships from the EMBO (ALTF 652-2019), Astellas Foundation for research on metabolic disorders, Mochida memorial foundation for medical and pharmaceutical research, ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), European Project: 771599,ICEBERG, European Project: 741773,AAA, Martin-Laffon, Jacqueline, CBH-EUR-GS - - CBH-EUR-GS2017 - ANR-17-EURE-0003 - EURE - VALID, Exploration below the tip of the microtubule - ICEBERG - 771599 - INCOMING, and Adaptive Actin Architectures - AAA - 741773 - INCOMING
Subjects: Centrosome, General Immunology and Microbiology, synthetic cell, General Neuroscience, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, centrosome positioning, Microtubules, General Biochemistry, Genetics and Molecular Biology, Actins, Actin Cytoskeleton, MTOC, actin network, Molecular Biology, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Microtubule-Organizing Center, microtubule
Abstract: International audience; The orientation of cell polarity depends on the position of the centrosome, the main microtubule-organizing center (MTOC). Microtubules (MTs) transmit pushing forces to the MTOC as they grow against the cell periphery. How the actin network regulates these forces remains unclear. Here, in a cell-free assay, we used purified proteins to reconstitute the interaction of a microtubule aster with actin networks of various architectures in cell-sized microwells. In the absence of actin filaments, MTOC positioning was highly sensitive to variations in microtubule length. The presence of a bulk actin network limited microtubule displacement, and MTOCs were held in place. In contrast, the assembly of a branched actin network along the well edges centered the MTOCs by maintaining an isotropic balance of pushing forces. An anisotropic peripheral actin network caused the MTOC to decenter by focusing the pushing forces. Overall, our results show that actin networks can limit the sensitivity of MTOC positioning to microtubule length and enforce robust MTOC centering or decentering depending on the isotropy of its architecture.
Published: 2022
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16. Author Reply to Peer Reviews of The architecture of the actin network can balance the pushing forces produced by growing microtubules

Author: Manuel Théry, Laurent Blanchoin, Magali Orhant-Prioux, Christophe Guerin, Benoit Vianay, Jérémie Gaillard, and Shohei Yamamoto
Published: 2022
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17. Visualization and Quantification of Microtubule Self-Repair

Author: Jérémie Gaillard, Laurent Blanchoin, Manuel Théry, Laura Schaedel, CytoMorphoLab, Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Ecotaxie, microenvironnement et développement lymphocytaire (EMily (UMR_S_1160 / U1160)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), and Faculty of Natural Sciences and Technology, Saarland University
Subjects: [SDV.BC]Life Sciences [q-bio]/Cellular Biology, ComputingMilieux_MISCELLANEOUS
Abstract: International audience
Published: 2022
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18. Visualization and Quantification of Microtubule Self-Repair

Author: Jérémie, Gaillard, Laurent, Blanchoin, Manuel, Théry, and Laura, Schaedel
Subjects: Tubulin, Microtubules
Abstract: Since its discovery, several decades ago, microtubule dynamic instability has been the subject of countless studies that demonstrate its impact on cellular behavior in health and disease. Recent studies reveal a new dimension of microtubule dynamics. Microtubules are not only dynamic at their tips but also exhibit loss and incorporation of tubulin subunits along their lattice far from the tips. Although this phenomenon has been observed to occur under various conditions in vitro as well as in cells, many questions remain regarding the regulation of lattice dynamics and their contribution to overall microtubule network organization and function. Compared to microtubule tip dynamics, the dynamics of tubulin incorporation along the lattice are more challenging to investigate as they are hidden in classical experimental setups, which is likely the reason they were overlooked for a long time. In this chapter, we present a strategy to visualize and quantify the incorporation of tubulin subunits into the microtubule lattice in vitro. The proposed method does not require specialized equipment and can thus be carried out readily in most research laboratories.
Published: 2022

19. Insights into the evolution of regulated actin dynamics via characterization of primitive gelsolin/cofilin proteins from Asgard archaea

Author: Edward Manser, Robert Robinson, Linh T. Tran, Yohendran Baskaran, Laurent Blanchoin, Magali Orhant-Prioux, Caner Akıl, Agency for science, technology and research [Singapore] (A*STAR), Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Research Institute for Interdisciplinary Science, Okayama University, CytoMorphoLab, Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Vidyasirimedhi Institute of Science and Technology, Agency for Science, Technology and Research, Singapore National Medical Research Council (NMRC Grant OFIRG/0067/2018), Vidyasirimedhi Institute of Science and Technology, Research Institute for Interdisciplinary Science, Japan Society for the Promotion of Science (KAKENHI Grant JP20H00476), European Project: 741773,AAA, Department of Pharmacology, Yong Loo Lin School of Medicine [Singapour], National University of Singapore (NUS), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), and Vidyasirimedhi Institute of Science and Technology [Thaïlande] (VISTEC)
Subjects: Protein Conformation, alpha-Helical, gelsolin, Archaeal Proteins, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, macromolecular substances, Biochemistry, Polymerization, Evolution, Molecular, 03 medical and health sciences, 0302 clinical medicine, Genome, Archaeal, Lokiarchaeota, Amino Acid Sequence, Cytoskeleton, Actin, X-ray crystallography, 030304 developmental biology, 0303 health sciences, Multidisciplinary, biology, Chemistry, Actin filament severing, Biological Sciences, eukaryogenesis, Cofilin, musculoskeletal system, Actin cytoskeleton, Archaea, Actins, Cell biology, Actin Depolymerizing Factors, Asgard archaea, Profilin, biology.protein, Sequence Alignment, actin, Filopodia, Gelsolin, 030217 neurology & neurosurgery
Abstract: Significance Eukaryotic gelsolin superfamily proteins generally comprise three or more related domains. Here we characterize single- and double-domain gelsolins from Thorarchaeota (Thor). Similar domain architectures are present in Heimdall-, Loki-, and Odinarchaeota. Thor gelsolins are functional in regulating rabbit actin in in vitro assays, showing a range of activities including actin filament severing and bundling. These gelsolins bind to the eukaryotic gelsolin/cofilin-binding site on actin. Two-domain, but not one-domain, gelsolins are calcium regulated. Thor gelsolins appear to have the characteristics and structure consistent with primitive gelsolins/cofilins, suggesting that these single- and double-domain gelsolins are a record of a nascent preeukaryotic actin-regulation machinery., Asgard archaea genomes contain potential eukaryotic-like genes that provide intriguing insight for the evolution of eukaryotes. The eukaryotic actin polymerization/depolymerization cycle is critical for providing force and structure in many processes, including membrane remodeling. In general, Asgard genomes encode two classes of actin-regulating proteins from sequence analysis, profilins and gelsolins. Asgard profilins were demonstrated to regulate actin filament nucleation. Here, we identify actin filament severing, capping, annealing and bundling, and monomer sequestration activities by gelsolin proteins from Thorarchaeota (Thor), which complete a eukaryotic-like actin depolymerization cycle, and indicate complex actin cytoskeleton regulation in Asgard organisms. Thor gelsolins have homologs in other Asgard archaea and comprise one or two copies of the prototypical gelsolin domain. This appears to be a record of an initial preeukaryotic gene duplication event, since eukaryotic gelsolins are generally comprise three to six domains. X-ray structures of these proteins in complex with mammalian actin revealed similar interactions to the first domain of human gelsolin or cofilin with actin. Asgard two-domain, but not one-domain, gelsolins contain calcium-binding sites, which is manifested in calcium-controlled activities. Expression of two-domain gelsolins in mammalian cells enhanced actin filament disassembly on ionomycin-triggered calcium release. This functional demonstration, at the cellular level, provides evidence for a calcium-controlled Asgard actin cytoskeleton, indicating that the calcium-regulated actin cytoskeleton predates eukaryotes. In eukaryotes, dynamic bundled actin filaments are responsible for shaping filopodia and microvilli. By correlation, we hypothesize that the formation of the protrusions observed from Lokiarchaeota cell bodies may involve the gelsolin-regulated actin structures.
Published: 2020
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20. Microtubules self-repair in living cells

Author: Morgan Gazzola, Alexandre Schaeffer, Benoit Vianay, Jérémie Gaillard, Laurent Blanchoin, and Manuel Théry
Abstract: Microtubule self-repair has been studied both in vitro and in vivo as an underlying mechanism of microtubule stability. The turnover of tubulin dimers along the microtubule network has challenged the pre-existing dogma that only growing ends are dynamic. However, although there is clear evidence of tubulin incorporation into the shaft of polymerized microtubules in vitro, the possibility of such events taking place in living cells remains uncertain. In this study, we investigated this possibility by microinjecting purified tubulin dimers labeled with a red fluorophore into the cytoplasm of cells expressing GFP-tubulin. We observed the appearance of red dots along pre-existing green microtubule network within minutes. We found that the fluorescence intensities of these red dots were inversely correlated with the green signal, suggesting that the red dimers were incorporated into the microtubules and replaced the pre-existing green dimers. We then characterized the size and spatial frequency of these incorporations as a function of injected tubulin concentration and post-injection delay. The saturation of these measurements contradicted the hypothesis of nonspecific adsorption along microtubules and suggested that the injected dimers incorporated into a finite number of damaged sites. By our low estimate, within a few minutes of the injections, free dimers incorporated into major repair sites every 70 micrometers of microtubules. Finally, we mapped the location of these sites in micropatterned cells and found that they were more concentrated in regions where the actin filament network was less dense and where microtubules exhibited greater lateral fluctuations. These results provide evidences that microtubules do self-repair in living cells, and they provide a quantitative characterization of the temporal and spatial dynamics of this process in PtK2 cells.
Published: 2022
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21. Cytoskeleton regulation: Distinct steps in Arp2/3 complex activation

Author: Alexandra, Colin, Laurent, Blanchoin, CytoMorphoLab, Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Département de Thérapie Cellulaire [Hôpital Saint-Louis - APHP], Hopital Saint-Louis [AP-HP] (AP-HP), and Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)
Subjects: Actin Cytoskeleton, macromolecular substances, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, biological phenomena, cell phenomena, and immunity, General Agricultural and Biological Sciences, Microtubules, Actin-Related Protein 2-3 Complex, Actins, Cytoskeleton, Article, General Biochemistry, Genetics and Molecular Biology
Abstract: Arp2/3 complex nucleates branched actin filaments important for processes like DNA repair, endocytosis and cellular motility. Multiple factors are required to activate branching nucleation by Arp2/3 complex, including a WASP family protein and a pre-existing actin filament. Activation is achieved through two major conformational changes—subunit flattening and movement into the short pitch conformation—that allow the actin related proteins within the complex (Arp2 and Arp3) to mimic filamentous actin subunits, thereby templating new filament assembly. Some models suggest these changes are concerted and stimulated cooperatively by WASP and actin filaments, but how Arp2/3 complex integrates signals from multiple factors to drive switch-like activation of branching nucleation has been unknown. Here we use biochemical assays to show that instead of a concerted mechanism, signal integration by Arp2/3 complex occurs via distinct and unconcerted conformational changes; WASP stimulates the short pitch arrangement of Arp2 and Arp3 while actin filaments trigger a different activation step. An engineered Arp2/3 complex that bypasses the need for WASP but not actin filaments in activation potently assembles isotropic actin networks but fails to assemble sustained force-producing actin networks in bead motility assays. The engineered complex, which is crosslinked into the short pitch conformation, fails to target nucleation to the surface of the bead, creating unproductive branching events that deplete unpolymerized actin and halt assembly. Together our data demonstrate the requirement for multi-factor signal integration by Arp2/3 complex and highlight the importance of both the WASP- and actin filament-mediated activation steps in assembly of functional actin networks.
Published: 2022
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22. Compressive forces stabilise microtubules in living cells

Author: Yuhui Li, Ondřej Kučera, Damien Cuvelier, David M. Rutkowski, Mathieu Deygas, Dipti Rai, Tonja Pavlovič, Filipe Nunes Vicente, Matthieu Piel, Gregory Giannone, Dimitrios Vavylonis, Anna Akhmanova, Laurent Blanchoin, and Manuel Théry
Abstract: Cell mechano-sensation and adaptation are supported by the actin network. The microtubule network is not considered to be directly sensitive to mechanical forces acting on a cell. However, recent studies on isolated microtubulesin vitrohave shown that bending forces have an impact on their structure, composition and lifespan, suggesting that, in a cellular context, microtubules may react to mechanical forces. We tested this hypothesis in living cells by subjecting them to cycles of compressive forces and found that microtubules became distorted, less dynamic and more stable. This mechano-stabilisation depends on CLASP2, which relocates from the end to the deformed shaft of microtubules. These results demonstrate that microtubules in living cells have mechano-responsive properties that allow them to resist and even counteract the forces to which they are subjected.
Published: 2022
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23. The architecture of the actin network can balance the pushing forces produced by growing microtubules

Author: Shohei Yamamoto, Jérémie Gaillard, Benoit Vianay, Christophe Guerin, Magali Orhant-Prioux, Laurent Blanchoin, and Manuel Théry
Subjects: macromolecular substances
Abstract: The position of centrosome, the main microtubule-organizing center (MTOC), is instrumental in the definition of cell polarity. It is defined by the balance of tension and pressure forces in the network of microtubules (MTs). As MTs polymerize against the cell periphery, pressure increases and produces pushing forces on the MTOC. How the mechanical interplay between MTs and the actin network is involved in the regulation of these forces remains poorly understood, in particular because its investigation is technically limited by the structural and biochemical complexity of the cell cytoplasm. Here, in a cell-free assay, we used purified proteins to reconstitute the interaction of an aster of dynamic MTs with actin networks of various compositions and architectures in cell-sized microwells. In the absence of actin filaments, the positioning of the MTOC was highly sensitive to variations in MT length. The presence of a bulk actin filament network limited MTs deformation and displacement, and MTOCs were hold in place. In contrast, the assembly of a dense and branched actin network along the edges of the wells centered the MTOCs by preventing MT slippage and thus maintaining an isotropic balance of pushing forces. In agreement with this, an asymmetric peripheral actin network caused the MTOC to decenter by creating an asymmetry in the pushing forces. Numerical simulations demonstrated that steric hindrance by actin networks, at the tip or along the entire length of MTs, can modulate MTOC positioning, as observed in the experiments. Overall, our results show that actin networks can limit the sensitivity of MTOC positioning to MT length and enforce robust MTOC centering or decentering depending on its architecture.
Published: 2022
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24. Reconstituting the Interaction Between Purified Nuclei and Microtubule Network

Author: Gökçe Agsu, Jérémie Gaillard, Bruno Cadot, Laurent Blanchoin, Emmanuelle Fabre, and Manuel Théry
Published: 2022
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25. Structure and dynamics of Odinarchaeota tubulin and the implications for eukaryotic microtubule evolution

Author: Caner Akıl, Samson Ali, Linh T. Tran, Jérémie Gaillard, Wenfei Li, Kenichi Hayashida, Mika Hirose, Takayuki Kato, Atsunori Oshima, Kosuke Fujishima, Laurent Blanchoin, Akihiro Narita, Robert C. Robinson, Earth-Life Science Institute [Tokyo] (ELSI), Tokyo Institute of Technology [Tokyo] (TITECH), Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), CytoMorphoLab, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), National Laboratory of Solid State Microstructures [Nanjing University] (LSSMS), Nanjing University (NJU), JST CREST, Japan, grant number JPMJCR19S5, Japan Society for the Promotion of Science (JSPS), grant number JP20H00476, Moore-Simons Project on the Origin of the Eukaryotic Cell, grant number GBMF9743, and ELSI-First Logic Astrobiology Donation Program
Subjects: Eukaryotic Cells, Multidisciplinary, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Tubulin, Eukaryota, Guanosine Triphosphate, Microtubules
Abstract: International audience; Tubulins are critical for the internal organization of eukaryotic cells, and understanding their emergence is an important question in eukaryogenesis. Asgard archaea are the closest known prokaryotic relatives to eukaryotes. Here, we elucidated the apo and nucleotide-bound x-ray structures of an Asgard tubulin from hydrothermal living Odinarchaeota (OdinTubulin). The guanosine 5′-triphosphate (GTP)–bound structure resembles a microtubule protofilament, with GTP bound between subunits, coordinating the “+” end subunit through a network of water molecules and unexpectedly by two cations. A water molecule is located suitable for GTP hydrolysis. Time course crystallography and electron microscopy revealed conformational changes on GTP hydrolysis. OdinTubulin forms tubules at high temperatures, with short curved protofilaments coiling around the tubule circumference, more similar to FtsZ, rather than running parallel to its length, as in microtubules. Thus, OdinTubulin represents an evolutionary stage intermediate between prokaryotic FtsZ and eukaryotic microtubule-forming tubulins.
Published: 2022
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26. Flagella-like beating of actin bundles driven by self-organized myosin waves

Author: Marie Pochitaloff, Martin Miranda, Mathieu Richard, Atitheb Chaiyasitdhi, Yasuharu Takagi, Wenxiang Cao, Enrique M. De La Cruz, James R. Sellers, Jean-François Joanny, Frank Jülicher, Laurent Blanchoin, Pascal Martin, Laboratoire Physico-Chimie Curie [Institut Curie] (PCC), Institut Curie [Paris]-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Max Planck Institute for the Physics of Complex Systems (MPI-PKS), Max-Planck-Gesellschaft, National Heart, Lung, and Blood Institute [Bethesda] (NHLBI), Yale University [New Haven], Collège de France - Chaire Matière molle et biophysique, Collège de France (CdF (institution)), Technische Universität Dresden = Dresden University of Technology (TU Dresden), CytoMorphoLab, Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Ecotaxie, microenvironnement et développement lymphocytaire (EMily (UMR_S_1160 / U1160)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), United States National Institutes of Health Grant R35-GM135656, ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), ANR-12-BSV5-0014,Contract,Nouveaux systèmes biomimétiques pour étudier la contractilité acto-myosine cellulaire(2012), ANR-21-CE30-0057,ActoMyoBeat,Auto-organisation d'un système acto-myosine minimal en faisceaux de filaments polaires ondulants périodiquement(2021), ANR-11-LABX-0038,CelTisPhyBio,Des cellules aux tissus: au croisement de la Physique et de la Biologie(2011), European Project: 741773,AAA, Martin-Laffon, Jacqueline, Développment d'une infrastructure française distribuée coordonnée - - France-BioImaging2010 - ANR-10-INBS-0004 - INBS - VALID, BLANC - Nouveaux systèmes biomimétiques pour étudier la contractilité acto-myosine cellulaire - - Contract2012 - ANR-12-BSV5-0014 - BLANC - VALID, Auto-organisation d'un système acto-myosine minimal en faisceaux de filaments polaires ondulants périodiquement - - ActoMyoBeat2021 - ANR-21-CE30-0057 - AAPG2021 - VALID, Des cellules aux tissus: au croisement de la Physique et de la Biologie - - CelTisPhyBio2011 - ANR-11-LABX-0038 - LABX - VALID, and Adaptive Actin Architectures - AAA - 741773 - INCOMING
Subjects: [PHYS.MECA.BIOM] Physics [physics]/Mechanics [physics]/Biomechanics [physics.med-ph], General Physics and Astronomy, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, [PHYS.MECA.BIOM]Physics [physics]/Mechanics [physics]/Biomechanics [physics.med-ph], [SDV.BC] Life Sciences [q-bio]/Cellular Biology
Abstract: A fait l'objet d'un communiqué de presse CNRS - Institut Curie, le 9 août 2022:https://www.cnrs.fr/fr/auto-assemblage-moleculaire-reproduisant-le-mouvement-ondulatoire-des-flagelles-responsables-de-la; International audience; Wave-like beating of eukaryotic cilia and flagella—threadlike protrusions found in many cells and microorganisms—is a classic example of spontaneous mechanical oscillations in biology. This type of self-organized active matter raises the question of the coordination mechanism between molecular motor activity and cytoskeletal filament bending. Here we show that in the presence of myosin motors, polymerizing actin filaments self-assemble into polar bundles that exhibit wave-like beating. Importantly, filament beating is associated with myosin density waves initiated at twice the frequency of the actin-bending waves. A theoretical description based on curvature control of motor binding to the filaments and of motor activity explains our observations in a regime of high internal friction. Overall, our results indicate that the binding of myosin to actin depends on the actin bundle shape, providing a feedback mechanism between the myosin activity and filament deformations for the self-organization of large motor filament assemblies.
Published: 2022
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27. A new perspective on microtubule dynamics: destruction by molecular motors and self-repair

Author: Laurent Blanchoin, Jérémie Gaillard, Sarah Triclin, Daisuke Inoue, Manuel Théry, CytoMorphoLab, Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Immunologie humaine, physiopathologie & immunothérapie (HIPI (UMR_S_976 / U976)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), European Project: 741773,AAA, European Project: 771599,ICEBERG, and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité)
Subjects: 0303 health sciences, Molecular motor, Microtubule dynamics, Chemistry, Perspective (graphical), Self repair, Transport, Microtubule, General Medicine, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Microtubules, 03 medical and health sciences, 0302 clinical medicine, Tubulin, Self-repair, Neuroscience, 030217 neurology & neurosurgery, Cytoskeleton, 030304 developmental biology
Abstract: Microtubules are dynamic polymers, permanently assembling and disassembling, that serve as tracks for intra-cellular transport by molecular motors. We recently found that the low energy of tubulin dimer interactions allows for spontaneous loss of tubulin dimers from the microtubule lattice [1]. This raised the possibility that the mechanical work produced by molecular motors as they move on microtubules can break dimer interactions and trigger microtubule disassembly. In a recent study, we tested this hypothesis by studying the interplay between microtubules and moving molecular motors in vitro [2]. Our results show that molecular motors can remove tubulin dimers from the lattice and rapidly destroy microtubules. We also found that dimer removal by motors was compensated by the insertion of free tubulin dimers into the microtubule lattice. This self-repair mechanism allows microtubules to survive the damage induced by molecular motors as they move along their tracks. Our study reveals the existence of coupling between the motion of molecular motors and the renewal of the microtubule lattice.Les microtubules sont des polymères dynamiques, s’assemblant et se désassemblant en permanence, qui servent de pistes pour le transport intracellulaire par des moteurs moléculaires. Nous avons récemment découvert que la faible énergie des interactions entre les dimères de tubuline permet la perte spontanée des dimères de tubuline le long d’un microtubule [1]. Le travail mécanique produit par les moteurs moléculaires lorsqu’ils se déplacent sur les microtubules pourrait donc rompre ces faibles interactions entre dimères et déclencher le désassemblage des microtubules. Dans une étude récente, nous avons testé cette hypothèse en étudiant l’interaction entre les microtubules et les moteurs moléculaires en mouvement in vitro [2]. Nos résultats montrent que les moteurs moléculaires peuvent retirer les dimères de tubuline du réseau et détruire rapidement les microtubules. Nous avons également constaté que l’élimination des dimères par les moteurs était compensée par l’insertion de dimères de tubuline libres dans le réseau de microtubules. Ce mécanisme d’autoréparation permet aux microtubules de survivre aux dommages induits par les moteurs moléculaires lors de leurs déplacements. Notre étude révèle donc l’existence d’un couplage entre le mouvement des moteurs moléculaires et le renouvellement du réseau de microtubules.
Published: 2021
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28. Structure and dynamics of Odinarchaeota tubulin and the implications for eukaryotic microtubule evolution

Author: Wenfei Li, Caner Akıl, Laurent Blanchoin, Akihiro Narita, Atsunori Oshima, Kosuke Fujishima, Linh T. Tran, Takayuki Kato, Mika Hirose, Samson Ali, Kenichi Hayashida, Robert Robinson, and Jérémie Gaillard
Subjects: Tubulin, Tubule, GTP', biology, Microtubule, Chemistry, Protein subunit, biology.protein, Biophysics, GTPase, biology.organism_classification, FtsZ, Archaea
Abstract: Tubulins are critical for the internal organization of eukaryotic cells, and understanding their emergence is an important question in eukaryogenesis. Asgard archaea are the closest known prokaryotic relatives to eukaryotes. Here, we elucidated the apo and nucleotide-bound X-ray structures of an Asgard tubulin from hydrothermal-living Odinarchaeota (OdinTubulin). The GTP-bound structure resembles a microtubule protofilament, with GTP bound between subunits, coordinating the “+” end subunit through a network of water molecules and unexpectedly by two cations. A water molecule is located suitable for GTP hydrolysis. Time course crystallography and electron microscopy revealed conformational changes on GTP hydrolysis. OdinTubulin forms tubules at high temperatures, with short curved protofilaments coiling around the tubule circumference, more similar to FtsZ, rather than running parallel to its length, as in microtubules. Thus, OdinTubulin represents an evolution intermediate between prokaryotic FtsZ and eukaryotic microtubule-forming tubulins.
Published: 2021
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29. The biochemical composition of the actomyosin network sets the magnitude of cellular traction forces

Author: Laurent Blanchoin, Laetitia Kurzawa, Timothée Vignaud, Somanna Kollimada, Fabrice Senger, Manuel Théry, CytoMorphoLab, Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Clinique de Chirurgie Digestive et Endocrinienne [CHU Nantes], Centre hospitalier universitaire de Nantes (CHU Nantes), Immunologie humaine, physiopathologie & immunothérapie (HIPI (UMR_S_976 / U976)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), ANR-14-CE11-0003,MaxForce,Maximiser la production de force: de la molécule au tissu(2014), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), European Project: 741773,AAA, European Project: 771599,ICEBERG, Martin-Laffon, Jacqueline, Appel à projets générique - Maximiser la production de force: de la molécule au tissu - - MaxForce2014 - ANR-14-CE11-0003 - Appel à projets générique - VALID, CBH-EUR-GS - - CBH-EUR-GS2017 - ANR-17-EURE-0003 - EURE - VALID, Adaptive Actin Architectures - AAA - 741773 - INCOMING, Exploration below the tip of the microtubule - ICEBERG - 771599 - INCOMING, and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP)
Subjects: Cell Culture Techniques, Retinal Pigment Epithelium, macromolecular substances, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Myosins, Focal adhesion, Protein filament, 03 medical and health sciences, 0302 clinical medicine, Traction, Myosin, Molecular motor, Cell Adhesion, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Humans, Actinin, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Cell adhesion, Cytoskeleton, Molecular Biology, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Actin, 030304 developmental biology, 0303 health sciences, Focal Adhesions, Microscopy, Cell Biology, Articles, Actomyosin, Vinculin, Actins, Biomechanical Phenomena, biology.protein, Biophysics, 030217 neurology & neurosurgery
Abstract: International audience; The regulation of cellular force production relies on the complex interplay between a well-conserved set of proteins of the cytoskeleton: actin, myosin, and α-actinin. Despite our deep knowledge of the role of these proteins in force production at the molecular scale, our understanding of the biochemical regulation of the magnitude of traction forces generated at the entire-cell level has been limited, notably by the technical challenge of measuring traction forces and the endogenous biochemical composition in the same cell. In this study, we developed an alternative Traction-Force Microscopy (TFM) assay, which used a combination of hydrogel micropatterning to define cell adhesion and shape and an intermediate fixation/immunolabeling step to characterize strain energies and the endogenous protein contents in single epithelial cells. Our results demonstrated that both the signal intensity and the area of the Focal Adhesion (FA)–associated protein vinculin showed a strong positive correlation with strain energy in mature FAs. Individual contents from actin filament and phospho-myosin displayed broader deviation in their linear relationship to strain energies. Instead, our quantitative analyzes demonstrated that their relative amount exhibited an optimum ratio of phospho-myosin to actin, allowing maximum force production by cells. By contrast, although no correlation was identified between individual α-actinin content and strain energy, the ratio of α-actinin to actin filaments was inversely related to strain energy. Hence, our results suggest that, in the cellular model studied, traction-force magnitude is dictated by the relative numbers of molecular motors and cross-linkers per actin filament, rather than the amounts of an individual component in the cytoskeletal network. This assay offers new perspectives to study in more detail the complex interplay between the endogenous biochemical composition of individual cells and the force they produce.
Published: 2021
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30. Kinesin-6 Klp9 orchestrates spindle elongation by regulating microtubule sliding and growth

Author: Carlos Kikuti, Manuel Théry, Anne Houdusse, Laurent Blanchoin, Liang Ji, Phong T. Tran, Matthieu Gélin, Lara Katharina Krüger, Biologie Cellulaire et Cancer, Institut Curie [Paris]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Immunologie humaine, physiopathologie & immunothérapie (HIPI (UMR_S_976 / U976)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), CytoMorphoLab, Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Department of Cell and Developmental Biology, University of Pennsylvania, University of Pennsylvania, Fondation ARC pour la Recherche sur le Cancer, INCA (Institut National du Cancer), Ligue Contre le Cancer, European Project: 771599,ICEBERG, Martin-Laffon, Jacqueline, Exploration below the tip of the microtubule - ICEBERG - 771599 - INCOMING, Compartimentation et dynamique cellulaires (CDC), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), University of Pennsylvania [Philadelphia], Centre de recherche de l'Institut Curie [Paris], Institut Curie [Paris], Pennsylvania State University (Penn State), and Penn State System
Subjects: 0301 basic medicine, Time Factors, [SDV]Life Sciences [q-bio], Kinesins, Microtubules, 0302 clinical medicine, Gene Expression Regulation, Fungal, cell biology, Biology (General), Anaphase, biology, Chemistry, General Neuroscience, Molecular Motor Proteins, General Medicine, Microtubule sliding, Kinesin-6, Cell biology, Meiosis, mitotic spindle, microtubule sliding, Kinesin, Medicine, Microtubule-Associated Proteins, Signal Transduction, Research Article, QH301-705.5, Science, microtubule polymerisation, Spindle Apparatus, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, General Biochemistry, Genetics and Molecular Biology, Spindle elongation, 03 medical and health sciences, Microtubule, Schizosaccharomyces, Mitosis, XMAP215, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, mitosis, General Immunology and Microbiology, Spindle apparatus, 030104 developmental biology, Tubulin, biology.protein, Schizosaccharomyces pombe Proteins, 030217 neurology & neurosurgery, S. pombe
Abstract: Mitotic spindle function depends on the precise regulation of microtubule dynamics and microtubule sliding. Throughout mitosis, both processes have to be orchestrated to establish and maintain spindle stability. We show that during anaphase B spindle elongation in Schizosaccharomyces pombe, the sliding motor Klp9 (kinesin-6) also promotes microtubule growth in vivo. In vitro, Klp9 can enhance and dampen microtubule growth, depending on the tubulin concentration. This indicates that the motor is able to promote and block tubulin subunit incorporation into the microtubule lattice in order to set a well-defined microtubule growth velocity. Moreover, Klp9 recruitment to spindle microtubules is dependent on its dephosphorylation mediated by XMAP215/Dis1, a microtubule polymerase, creating a link between the regulation of spindle length and spindle elongation velocity. Collectively, we unravel the mechanism of anaphase B, from Klp9 recruitment to the motors dual-function in regulating microtubule sliding and microtubule growth, allowing an inherent coordination of both processes.
Published: 2021
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31. MICAL2 enhances branched actin network disassembly by oxidizing Arp3B-containing Arp2/3 complexes

Author: Christophe Guérin, Pavithra Singaravelu, Laurent Blanchoin, Naoko Kogata, Svend Kjaer, Davide Carra, Michael Way, Chiara Galloni, Jasmine V. Abella, David J. Barry, The Francis Crick Institute, Cellular Signalling and Cytoskeletal Function Laboratory, The Francis Crick Institute, Structural Biology Science Technology Platform, CytoMorphoLab, Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), CytoMorphoLab UMR976 HIPI, CEA, INSERM, Université de Paris, Paris, France, The Francis Crick Institute, Advanced Light Microscopy Facility, Imperial College, Department of infectious diseases, Cancer Research UK (FC001209), UK Medical Research Council (FC001209), Wellcome Trust (FC001209) funding at the Francis Crick Institute, European Project: Grant 810207, European Project: 741773,AAA, Martin-Laffon, Jacqueline, European Union’s Horizon 2020 research and innovation programme (grant 810207) - Grant 810207 - INCOMING, Adaptive Actin Architectures - AAA - 741773 - INCOMING, Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Gene isoform, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, macromolecular substances, Biochemistry, Article, Actin-Related Protein 2-3 Complex, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Oxidizing agent, Threonine, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Actin, Cytoskeleton, 030304 developmental biology, 0303 health sciences, Methionine, biology, Cell Biology, Monooxygenase, Actins, Glutamine, Actin Cytoskeleton, chemistry, biology.protein, Biophysics, 030217 neurology & neurosurgery, Cortactin
Abstract: Galloni, Carra, et al. demonstrate that Arp3B isoform–specific Arp2/3 complexes generate branched actin networks with faster disassembly kinetics. This increased turnover is due to oxidation of Met293 of Arp3B by the methionine monooxygenase MICAL2, which is recruited to the branched actin network by coronin 1C., The mechanisms regulating the disassembly of branched actin networks formed by the Arp2/3 complex still remain to be fully elucidated. In addition, the impact of Arp3 isoforms on the properties of Arp2/3 are also unexplored. We now demonstrate that Arp3 and Arp3B isocomplexes promote actin assembly equally efficiently but generate branched actin networks with different disassembly rates. Arp3B dissociates significantly faster than Arp3 from the network, and its depletion increases actin stability. This difference is due to the oxidation of Arp3B, but not Arp3, by the methionine monooxygenase MICAL2, which is recruited to the actin network by coronin 1C. Substitution of Arp3B Met293 by threonine, the corresponding residue in Arp3, increases actin network stability. Conversely, replacing Arp3 Thr293 with glutamine to mimic Met oxidation promotes disassembly. The ability of MICAL2 to enhance network disassembly also depends on cortactin. Our observations demonstrate that coronin 1C, cortactin, and MICAL2 act together to promote disassembly of branched actin networks by oxidizing Arp3B-containing Arp2/3 complexes.
Published: 2021
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32. Self-repair protects microtubules from destruction by molecular motors

Author: Samara L. Reck-Peterson, Sarah Triclin, Emmanuel Derivery, Jérémie Gaillard, Laurent Blanchoin, Morgan E. DeSantis, Laura Schaedel, Karin John, Zaw Min Htet, Didier Portran, Charlotte Aumeier, Christophe Leterrier, Manuel Théry, Daisuke Inoue, CytoMorphoLab, Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Kyushu University [Fukuoka], University of California [San Diego] (UC San Diego), University of California, Centre de recherche en Biologie Cellulaire (CRBM), Université Montpellier 1 (UM1)-Université Montpellier 2 - Sciences et Techniques (UM2)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), MRC Laboratory of Molecular Biology [Cambridge, UK] (LMB), University of Cambridge [UK] (CAM)-Medical Research Council, Université de Genève (UNIGE), Laboratoire Interdisciplinaire de Physique [Saint Martin d’Hères] (LIPhy ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Institut de neurophysiopathologie (INP), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Janelia Research Campus [Ashburn] (HHMI Janelia), Howard Hughes Medical Institute (HHMI), Immunologie humaine, physiopathologie & immunothérapie (HIPI (UMR_S_976 / U976)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Howard Hughes Medical Institute, IRTELIS PhD programme from the CEA, ANR-18-CE13-0001,APERTuRe,Une nouvelle perspective sur la régulation des microtubules(2018), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), European Project: 741773,AAA, European Project: 771599,ICEBERG, Martin-Laffon, Jacqueline, APPEL À PROJETS GÉNÉRIQUE 2018 - Une nouvelle perspective sur la régulation des microtubules - - APERTuRe2018 - ANR-18-CE13-0001 - AAPG2018 - VALID, Grenoble Alliance for Integrated Structural Cell Biology - - GRAL2010 - ANR-10-LABX-0049 - LABX - VALID, CBH-EUR-GS - - CBH-EUR-GS2017 - ANR-17-EURE-0003 - EURE - VALID, Adaptive Actin Architectures - AAA - 741773 - INCOMING, Exploration below the tip of the microtubule - ICEBERG - 771599 - INCOMING, Kyushu University, University of California (UC), Centre de recherche en Biologie cellulaire de Montpellier (CRBM), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Université de Genève = University of Geneva (UNIGE), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité)
Subjects: Microtubule disassembly, Movement, Dimer, 02 engineering and technology, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, 010402 general chemistry, Microtubules, Models, Biological, 01 natural sciences, Article, chemistry.chemical_compound, Microtubule, ATP hydrolysis, Molecular motor, General Materials Science, [PHYS.MECA.BIOM]Physics [physics]/Mechanics [physics]/Biomechanics [physics.med-ph], [SDV.BC] Life Sciences [q-bio]/Cellular Biology, biology, Chemistry, Molecular Motor Proteins, Mechanical Engineering, Self repair, [PHYS.MECA.BIOM] Physics [physics]/Mechanics [physics]/Biomechanics [physics.med-ph], General Chemistry, 021001 nanoscience & nanotechnology, Condensed Matter Physics, 0104 chemical sciences, Coupling (electronics), Tubulin, Mechanics of Materials, ddc:540, biology.protein, Biophysics, 0210 nano-technology
Abstract: International audience; Microtubule instability stems from the low energy of tubulin dimer interactions, which sets the growing polymer close to its disassembly conditions. Molecular motors use ATP hydrolysis to produce mechanical work and move on microtubules. This raises the possibility that the mechanical work produced by walking motors can break dimer interactions and trigger microtubule disassembly. We tested this hypothesis by studying the interplay between microtubules and moving molecular motors in vitro. Our results show that molecular motors can remove tubulin dimers from the lattice and rapidly destroy microtubules. We also found that dimer removal by motors was compensated for by the insertion of free tubulin dimers into the microtubule lattice. This self-repair mechanism allows microtubules to survive the damage induced by molecular motors as they move along their tracks. Our study reveals the existence of coupling between the motion of molecular motors and the renewal of the microtubule lattice.
Published: 2021
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33. Author response: Kinesin-6 Klp9 orchestrates spindle elongation by regulating microtubule sliding and growth

Author: Liang Ji, Anne Houdusse, Carlos Kikuti, Phong T. Tran, Lara Katharina Krüger, Manuel Théry, Laurent Blanchoin, and Matthieu Gélin
Subjects: Chemistry, Kinesin, Microtubule sliding, Spindle elongation, Cell biology
Published: 2021
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34. Stress fibres are embedded in a contractile cortical network

Author: Christophe Leterrier, Laurent Blanchoin, Calina Copos, Qingzong Tseng, Julia Mahamid, Timothée Vignaud, Mauricio Toro-Nahuelpan, Laetitia Kurzawa, Manuel Théry, Alex Mogilner, CytoMorphoLab, Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Clinique de Chirurgie Digestive et Endocrinienne [CHU Nantes], Centre hospitalier universitaire de Nantes (CHU Nantes), Courant Institute and Department of Biology, New York University [New York] (NYU), NYU System (NYU)-NYU System (NYU), Institut de neurophysiopathologie (INP), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany, US Army Research Office (grant W911NF-17-1-0417), European Molecular Biology Laboratory (EMBL), Marie Skłodowska-Curie Actions COFUND (664726), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-14-CE11-0003,MaxForce,Maximiser la production de force: de la molécule au tissu(2014), European Project: 741773,AAA, European Project: 771599,ICEBERG, Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)
Subjects: Materials science, [SDV]Life Sciences [q-bio], Traction (engineering), Photoablation, 02 engineering and technology, Retinal Pigment Epithelium, macromolecular substances, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, 010402 general chemistry, Microscopy, Atomic Force, 01 natural sciences, Traction force microscopy, Models, Biological, Article, Cell Line, Elastic Modulus, Stress Fibers, Humans, General Materials Science, Actin, Coalescence (physics), Mechanical Engineering, Mechanical impact, Cryoelectron Microscopy, Hydrogels, General Chemistry, 021001 nanoscience & nanotechnology, Condensed Matter Physics, Actins, 0104 chemical sciences, Biomechanical Phenomena, Actin Cytoskeleton, Mechanics of Materials, Cortical network, Biophysics, 0210 nano-technology, Micropatterning
Abstract: International audience; Contractile actomyosin networks are responsible for the production of intracellular forces. There is increasing evidence that bundles of actin filaments form interconnected and interconvertible structures with the rest of the network. In this study, we explored the mechanical impact of these interconnections on the production and distribution of traction forces throughout the cell. By using a combination of hydrogel micropatterning, traction force microscopy and laser photoablation, we measured the relaxation of traction forces in response to local photoablations. Our experimental results and modelling of the mechanical response of the network revealed that bundles were fully embedded along their entire length in a continuous and contractile network of cortical filaments. Moreover, the propagation of the contraction of these bundles throughout the entire cell was dependent on this embedding. In addition, these bundles appeared to originate from the alignment and coalescence of thin and unattached cortical actin filaments from the surrounding mesh.
Published: 2021
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35. Kinesin-6 Klp9 orchestrates spindle elongation by regulating microtubule sliding and growth

Author: Anne Houdusse, Laurent Blanchoin, Carlos Kikuti, Phong T. Tran, Liang Ji, Lara Katharina Krüger, Manuel Théry, and Matthieu Gélin
Subjects: Tubulin, biology, Microtubule, Chemistry, biology.protein, Kinesin, Microtubule sliding, Mitosis, Spindle elongation, Spindle apparatus, Cell biology, Anaphase
Abstract: Mitotic spindle function depends on the precise regulation of microtubule dynamics and microtubule sliding. Throughout mitosis, both processes have to be orchestrated to establish and maintain spindle stability. We show that during anaphase B spindle elongation inS. pombe, the sliding motor Klp9 (kinesin-6) also promotes microtubule growthin vivo. In vitro, Klp9 can enhance and dampen microtubule growth, depending on the tubulin concentration. This indicates that the motor is able to promote and block tubulin subunit incorporation into the microtubule lattice in order to set a well-defined microtubule growth velocity. Moreover, Klp9 recruitment to spindle microtubules is dependent on its dephosphorylation mediated by XMAP215/Dis1, a microtubule polymerase, to link the regulation of spindle length and spindle elongation velocity. Collectively, we unravel the mechanism of anaphase B, from Klp9 recruitment to the motors dual-function in regulating microtubule sliding and microtubule growth, allowing an inherent coordination of both processes.
Published: 2021
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36. Hematopoietic progenitors polarize in contact with bone marrow stromal cells in response to SDF1

Author: Khansa Saadallah, Benoit Vianay, Stephane Brunet, Jérôme Larghero, Adrian Candelas, Alexandre Schaeffer, Laurent Blanchoin, Samy Gobaa, Thierry Jaffredo, Damien Cuvelier, Julien Lion, Nuala Mooney, Thomas Bessy, Lionel Faivre, Jean-Christophe Bories, Manuel Thery, Cecilia Nakid-Cordero, Benoit Souquet, CytoMorphoLab, Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Institut Pierre-Gilles de Gennes pour la Microfluidique, Biologie Cellulaire et Cancer, Institut Curie [Paris]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Plateforme technologique Biomatériaux et Microfluidique - Biomaterials and Microfluidics technologic Platform, Institut Pasteur [Paris], Immunologie humaine, physiopathologie & immunothérapie (HIPI (UMR_S_976 / U976)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Laboratoire de Biologie du Développement [Paris] (LBD), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), This work was funded by grants from the Agence Nationale de la Recherche (ANR-14-CE11-0012, ANR-10-IHUB-0002), the European Research Council (ERC CoG 771599), the Emergence program of the Ville de Paris, the 'Coups d’Elan' prize of the Fondation Bettencourt Schueller, and the Fondation Schlumberger pour l’Education et la Recherche. T. Bessy received a PhD fellowship from the Université de Paris and the Ligue Contre le Cancer. The facility is supported by the Conseil Régional, Île-de-France, Canceropôle Ile-de-France, Université de Paris, Association Saint-Louis, Association Jean-Bernard, Fondation pour la Recherche Médicale, the French Institut National Du Cancer, and Ministère de la Recherche., ANR-14-CE11-0012,STAR,Architectures des cellules souches : Rôle des interactions cellules souches hematopoïétiques et mésenchymateuses dans le contrôle de leur polarisation et de l'asymétrie de leurs divisions cellulaires.(2014), ANR-10-IBHU-0002,SLI,Institut Saint-Louis(2010), European Project: 771599,ICEBERG, Institut Pasteur [Paris] (IP), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Laboratoire de Biologie du Développement [IBPS] (LBD), Mooney, Nuala, Appel à projets générique - Architectures des cellules souches : Rôle des interactions cellules souches hematopoïétiques et mésenchymateuses dans le contrôle de leur polarisation et de l'asymétrie de leurs divisions cellulaires. - - STAR2014 - ANR-14-CE11-0012 - Appel à projets générique - VALID, Instituts Hospitalo-Universitaires B - Institut Saint-Louis - - SLI2010 - ANR-10-IBHU-0002 - IBHU - VALID, and Exploration below the tip of the microtubule - ICEBERG - 771599 - INCOMING
Subjects: Stromal cell, [SDV.BC.IC] Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Biology, 03 medical and health sciences, 0302 clinical medicine, Bone Marrow, Microtubule, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], medicine, Humans, Progenitor cell, Cytoskeleton, Cells, Cultured, 030304 developmental biology, 0303 health sciences, Endothelial Cells, Mesenchymal Stem Cells, Cell Biology, Hematopoietic Stem Cells, Chemokine CXCL12, Cell biology, Haematopoiesis, medicine.anatomical_structure, Centrosome, 030220 oncology & carcinogenesis, Bone marrow, Stem cell
Abstract: This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).; International audience; The fate of hematopoietic stem and progenitor cells (HSPCs) is regulated by their interaction with stromal cells in the bone marrow. However, the cellular mechanisms regulating HSPC interaction with these cells and their potential impact on HSPC polarity are still poorly understood. Here we evaluated the impact of cell–cell contacts with osteoblasts or endothelial cells on the polarity of HSPC. We found that an HSPC can form a discrete contact site that leads to the extensive polarization of its cytoskeleton architecture. Notably, the centrosome was located in proximity to the contact site. The capacity of HSPCs to polarize in contact with stromal cells of the bone marrow appeared to be specific, as it was not observed in primary lymphoid or myeloid cells or in HSPCs in contact with skin fibroblasts. The receptors ICAM, VCAM, and SDF1 were identified in the polarizing contact. Only SDF1 was independently capable of inducing the polarization of the centrosome–microtubule network.
Published: 2021
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37. Microtubules control nuclear shape and gene expression during early stages of hematopoietic differentiation

Author: Lionel Faivre, Stéphane Brunet, Gökçe Agsu, Manuel Théry, Laurent Blanchoin, Benoit Vianay, Stefan Biedzinski, Marc Delord, Jérôme Larghero, CytoMorphoLab, Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Immunologie humaine, physiopathologie & immunothérapie (HIPI (UMR_S_976 / U976)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Centre Hospitalier de Versailles, Recherche Clinique et Investigation, Département de Thérapie Cellulaire [Hôpital Saint-Louis - APHP], Hopital Saint-Louis [AP-HP] (AP-HP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Emergence program of the Ville de Paris, 'Coups d’Elan' prize of the Bettencourt-Schueller foundation, Schlumberger foundation for education and research, ANR-14-CE11-0012,STAR,Architectures des cellules souches : Rôle des interactions cellules souches hematopoïétiques et mésenchymateuses dans le contrôle de leur polarisation et de l'asymétrie de leurs divisions cellulaires.(2014), European Project: 771599,ICEBERG, and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité)
Subjects: Myeloid, Lineage (genetic), Heterochromatin, Gene Expression, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Microtubules, General Biochemistry, Genetics and Molecular Biology, Chromatin remodeling, Cell Line, chromatin remodeling, Histones, 03 medical and health sciences, 0302 clinical medicine, stem cells, Gene expression, medicine, Humans, Cell Lineage, News & Views, Progenitor cell, Molecular Biology, nucleus deformation, 030304 developmental biology, Cell Nucleus, 0303 health sciences, General Immunology and Microbiology, General Neuroscience, Cell Differentiation, Articles, differentiation, Hematopoietic Stem Cells, Hematopoiesis, Cell biology, Haematopoiesis, medicine.anatomical_structure, Cytokines, sense organs, Stem cell, Transcriptome, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, 030217 neurology & neurosurgery, microtubule
Abstract: International audience; Hematopoietic stem and progenitor cells (HSPC) can differentiate into all hematopoietic lineages to support hematopoiesis. Cells from the myeloid and lymphoid lineages fulfill distinct functions with specific shapes and intra-cellular architectures. The role of cytokines in the regulation of HSPC differentiation has been intensively studied but our understanding of the potential contribution of inner cell architecture is relatively poor. Here, we show that large invaginations are generated by microtubule constraints on the swelling nucleus of human HSPC during early commitment toward the myeloid lineage. These invaginations are associated with a local reduction of lamin B density, local loss of heterochromatin H3K9me3 and H3K27me3 marks, and changes in expression of specific hematopoietic genes. This establishes the role of microtubules in defining the unique lobulated nuclear shape observed in myeloid progenitor cells and suggests that this shape is important to establish the gene expression profile specific to this hematopoietic lineage. It opens new perspectives on the implications of microtubule-generated forces, in the early commitment to the myeloid lineage.
Published: 2020
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38. MICAL2 acts through Arp3B isoform-specific Arp2/3 complexes to destabilize branched actin networks

Author: Svend Kjaer, Laurent Blanchoin, Davide Carra, Michael Way, David J. Barry, Jasmine V. Abella, Pavithra Singaravelu, Chiara Galloni, Naoko Kogata, and Christophe Guérin
Subjects: Gene isoform, Protein filament, chemistry.chemical_compound, Residue (chemistry), Methionine, Chemistry, Biophysics, macromolecular substances, Monooxygenase, Actin, In vitro
Abstract: The Arp2/3 complex (Arp2, Arp3 and ARPC1-5) is essential to generate branched actin filament networks for many cellular processes. Human Arp3, ARPC1 and ARPC5 exist as two isoforms but the functional properties of Arp2/3 iso-complexes is largely unexplored. Here we show that Arp3B, but not Arp3 is subject to regulation by the methionine monooxygenase MICAL2, which is recruited to branched actin networks by coronin-1C. Although Arp3 and Arp3B iso-complexes promote actin assembly equally efficiently in vitro, they have different cellular properties. Arp3B turns over significantly faster than Arp3 within the network and upon its depletion actin turnover decreases. Substitution of Arp3B Met293 by Thr, the corresponding residue in Arp3 increases actin network stability, and conversely, replacing Arp3 Thr293 with Gln to mimic Met oxidation promotes network disassembly. Thus, MICAL2 regulates a subset of Arp2/3 complexes to control branched actin network disassembly.
Published: 2020
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39. Hematopoietic progenitors polarize in contact with bone marrow stromal cells by engaging CXCR4 receptors

Author: Laurent Blanchoin, Alexandre Schaeffer, Stéphane Brunet, Thomas Bessy, Thierry Jaffredo, Benoit Vianay, Benoit Souquet, Lionel Faivre, Manuel Théry, and Jérôme Larghero
Subjects: 0303 health sciences, Stromal cell, Chemistry, CXCR4, Cell biology, 03 medical and health sciences, Haematopoiesis, 0302 clinical medicine, medicine.anatomical_structure, Microtubule, Centrosome, 030220 oncology & carcinogenesis, medicine, Bone marrow, Progenitor cell, Cytoskeleton, 030304 developmental biology
Abstract: Hematopoietic stem and progenitor cells (HSPCs) are located in the bone marrow, where they regulate the permanent production and renewal of all blood-cell types. HSPC proliferation and differentiation is locally regulated by their interaction with cells forming specific microenvironments close to the bone matrix or close to blood vessels. However, the cellular mechanisms underlying HSPC’s interaction with these cells and their potential impact on HSPC polarity is still poorly understood. Here we modelled the bone-marrow niche using microfluidic technologies in a bone-marrow on a chip device, and evaluated long-duration cell-cell contacts between single HSPCs and stromal cells or endothelial cells in a custom-designed microwell cell-culture system. We found that an HSPC can form a discrete contact site that leads to the extensive polarization of their cytoskeleton architectures. As in the case with immune synapses formed by lymphocytes, the centrosome was located in proximity of the cell-cell contact. The entire microtubule network emanated from the centrosome, and the nucleus was confined to the side opposite of the cell-cell contact. The capacity of the HSPC to polarize appeared specific as it was not observed in contact with skin fibroblasts. The receptors ICAM, VCAM and CXCR4 were identified in the polarizing contact, and were all independently capable of inducing morphological polarization. However, only CXCR4 was independently capable of inducing the polarization of the centrosome-microtubule network. Altogether these results revealed a novel mechanism of HSPC polarization associated with its anchorage to specific cells in the bone-marrow, which might be instrumental in the regulation of their fate.
Published: 2020
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40. Loss of Ena/VASP interferes with lamellipodium architecture, motility and integrin-dependent adhesion

Author: Georgi Dimchev, Michael Sixt, Matthias Schaks, Joern Linkner, Stefan Bruehmann, Laurent Blanchoin, Maria Nemethova, Klemens Rottner, Laetitia Kurzawa, Jan Mueller, Thomas Pokrant, Julia Damiano-Guercio, Jan Faix, Institute for Biophysical Chemistry, Hannover Medical School [Hannover] (MHH), CytoMorphoLab, Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institute of Science and Technology [Austria] (IST Austria), Technical University Braunschweig, Helmholtz Centre for Infection Research, Deutsche Forschungsgemeinschaft (DFG), grants FA330/11-1 and RO2414/5-1, PROCOMPAS graduate program GRK2223/1, European Project: 741773,AAA, European Project: CoG 724373,GRADIENTSENSING, Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Institute of Science and Technology [Klosterneuburg, Austria] (IST Austria), and HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Subjects: Integrins, cell migration, Melanoma, Experimental, Polymerization, Gene Knockout Techniques, Mice, 0302 clinical medicine, Cell Movement, cell biology, microspikes, Pseudopodia, Biology (General), Cytoskeleton, 0303 health sciences, biology, Chemistry, General Neuroscience, cytoskeleton, Cell migration, General Medicine, Recombinant Proteins, Cell biology, DNA-Binding Proteins, Actin Cytoskeleton, Medicine, Lamellipodium, Filopodia, Research Article, QH301-705.5, Science, Actin Capping Proteins, Integrin, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, macromolecular substances, Actin-Related Protein 2-3 Complex, General Biochemistry, Genetics and Molecular Biology, Focal adhesion, 03 medical and health sciences, filopodia, Cell Line, Tumor, Animals, Cell adhesion, mouse, Actin, 030304 developmental biology, Focal Adhesions, General Immunology and Microbiology, Ena/VASP proteins, cell adhesion, Fibroblasts, Actins, lamellipodia, NIH 3T3 Cells, biology.protein, CRISPR-Cas Systems, Actin filaments, 030217 neurology & neurosurgery
Abstract: International audience; Cell migration entails networks and bundles of actin filaments termed lamellipodia and microspikes or filopodia, respectively, as well as focal adhesions, all of which recruit Ena/VASP family members hitherto thought to antagonize efficient cell motility. However, we find these proteins to act as positive regulators of migration in different murine cell lines. CRISPR/Cas9-mediated loss of Ena/VASP proteins reduced lamellipodial actin assembly and perturbed lamellipodial architecture, as evidenced by changed network geometry as well as reduction of filament length and number that was accompanied by abnormal Arp2/3 complex and heterodimeric capping protein accumulation. Loss of Ena/VASP function also abolished the formation of microspikes normally embedded in lamellipodia, but not of filopodia capable of emanating without lamellipodia. Ena/VASP-deficiency also impaired integrin-mediated adhesion accompanied by reduced traction forces exerted through these structures. Our data thus uncover novel Ena/VASP functions of these actin polymerases that are fully consistent with their promotion of cell migration.
Published: 2020
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41. Author response: Loss of Ena/VASP interferes with lamellipodium architecture, motility and integrin-dependent adhesion

Author: Maria Nemethova, Klemens Rottner, Laetitia Kurzawa, Laurent Blanchoin, Matthias Schaks, Jan Mueller, Georgi Dimchev, Julia Damiano-Guercio, Michael Sixt, Joern Linkner, Thomas Pokrant, Stefan Brühmann, and Jan Faix
Subjects: biology, Chemistry, Integrin, biology.protein, Motility, Adhesion, Lamellipodium, Cell biology
Published: 2020
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42. Stress fibers are embedded in a contractile cortical network

Author: Qingzong Tseng, Laurent Blanchoin, Laetitia Kurzawa, Christophe Leterrier, Calina Copos, Timothée Vignaud, Alex Mogilner, and Manuel Théry
Subjects: 0303 health sciences, Materials science, Mechanical impact, Photoablation, Extracellular matrix, 03 medical and health sciences, 0302 clinical medicine, Cortical network, Biophysics, Cell shape, 030217 neurology & neurosurgery, Actin, Intracellular, 030304 developmental biology, Micropatterning
Abstract: Contractile actomyosin networks generate intracellular forces essential for the regulation of cell shape, migration, and cell-fate decisions, ultimately leading to the remodeling and patterning of tissues. Although actin filaments aligned in bundles represent the main source of traction-force production in adherent cells, there is increasing evidence that these bundles form interconnected and interconvertible structures with the rest of the intracellular actin network. In this study, we explored how these bundles are connected to the surrounding cortical network and the mechanical impact of these interconnected structures on the production and distribution of traction forces on the extracellular matrix and throughout the cell. By using a combination of hydrogel micropatterning, traction-force microscopy and laser photoablation, we measured the relaxation of the cellular traction field in response to local photoablations at various positions within the cell. Our experimental results and modeling of the mechanical response of the network revealed that bundles were fully embedded along their entire length in a continuous and contractile network of cortical filaments. Moreover, the propagation of the contraction of these bundles throughout the entire cell was dependent on this embedding. In addition, these bundles appeared to originate from the alignment and coalescence of thin and unattached cortical actin filaments from the surrounding mesh.
Published: 2020
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43. Loss of Ena/VASP interferes with lamellipodium architecture, motility and integrin-dependent adhesion

Author: Klemens Rottner, Jan Faix, Stefan Bruehmann, Laetitia Kurzawa, Thomas Pokrant, Michael Sixt, Georgi Dimchev, Julia Damiano-Guercio, Joern Linkner, Jan Mueller, Laurent Blanchoin, and Maria Nemethova
Subjects: Focal adhesion, Protein filament, biology, Chemistry, Integrin, biology.protein, Motility, Cell migration, macromolecular substances, Lamellipodium, Filopodia, Actin, Cell biology
Abstract: Cell migration entails networks and bundles of actin filaments termed lamellipodia and microspikes or filopodia, respectively, as well as focal adhesions, all of which recruit Ena/VASP family members hitherto thought to antagonize efficient cell motility. However, we find these proteins to act as positive regulators of migration in different cell lines. CRISPR/Cas9-mediated loss of Ena/VASP proteins reduced lamellipodial actin assembly and perturbed lamellipodial architecture, as evidenced by changed network geometry as well as reduction of filament length and number that was accompanied by abnormal Arp2/3 complex and heterodimeric capping protein accumulation. Loss of Ena/VASP function also abolished the formation of microspikes normally embedded in lamellipodia, but not of filopodia capable of emanating without lamellipodia. Ena/VASP-deficiency also impaired integrin-mediated adhesion accompanied by reduced traction forces exerted through these structures. Our data thus uncover novel, cellular Ena/VASP functions of these actin polymerases that are fully consistent with their promotion of cell migration.
Published: 2020
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44. Tailoring cryo-electron microscopy grids by photo-micropatterning for in-cell structural studies

Author: Laurent Blanchoin, Manuel Théry, Julia Mahamid, Fabrice Senger, Mauricio Toro-Nahuelpan, Ievgeniia Zagoriy, European Molecular Biology Laboratory, Structural and Computational Biology Unit, Heidelberg, Germany, CytoMorphoLab, Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Physiologie cellulaire et végétale (LPCV), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut National de la Recherche Agronomique (INRA), EMBL Interdisciplinary (EI3POD) program under Marie Skłodowska-Curie Actions COFUND (no. 664726), European Project: 760067,3DCellPhase, European Project: 771599,ICEBERG, European Project: 741773,AAA, Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
Subjects: Materials science, Ion beam, Cryo-electron microscopy, Green Fluorescent Proteins, Nanotechnology, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biochemistry, Focused ion beam, Article, Pattern Recognition, Automated, Specimen Handling, law.invention, 03 medical and health sciences, 0302 clinical medicine, law, Image Processing, Computer-Assisted, Humans, Cell adhesion, Molecular Biology, 030304 developmental biology, 0303 health sciences, Cell Membrane, Cryoelectron Microscopy, Molecular biophysics, Cell Biology, Adhesion, Molecular Imaging, Characterization (materials science), Surface micromachining, Electron microscope, 030217 neurology & neurosurgery, Intracellular, HeLa Cells, Biotechnology, Micropatterning
Abstract: Micromachining by cryo-focused ion beam (FIB) milling coupled to cryo-electron tomography (ET) enables visualization of macromolecules directly inside cells. Yet, spatial control of cell adhesion on electron microscopy (EM) grids remains a bottleneck in the specimen preparation pipeline. This protocol describes a contactless and mask-free photo-micropatterning of EM grids for site-specific deposition of extracellular matrix-related proteins. We achieved accurate and reproducible cell positioning, leading to optimized preparations for cryo-FIB milling. We tested HeLa and RPE1 cell lines on various grid types (gold or titanium mesh coated with SiO2, gold or carbon films). Briefly, grids were passivated with an anti-fouling agent, followed by controlled ablation of the passivation layer, and further functionalization with fibronectin. The micropatterning procedure takes ~3 h. Employing micropatterning to produce complex shapes generated a predictable intracellular organization, allowing direct correlation between cellular architecture and in-cell 3D-structural characterization of the underlying machinery at molecular resolution.
Published: 2020
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45. Force Production by a Bundle of Growing Actin Filaments Is Limited by Its Mechanical Properties

Author: Laurent Blanchoin, Jean Louis Martiel, Julien Berro, Alphée Michelot, Rajaa Boujemaa-Paterski, CytoMorphoLab, Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Physiologie cellulaire et végétale (LPCV), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Dynamique Cellulaire et Tissulaire- Interdisciplinarité, Modèles & Microscopies (TIMC-IMAG-DyCTiM), Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications, Grenoble - UMR 5525 (TIMC-IMAG), Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP)-IMAG-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP)-IMAG-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Institut de Biologie du Développement de Marseille (IBDM), Aix Marseille Université (AMU)-Collège de France (CdF)-Centre National de la Recherche Scientifique (CNRS), Department of Biochemistry - University of Zurich, Yale University [New Haven], National Institutes of Health/National Institute of General Medical Sciences Grant R01GM115636, ANR-14-CE11-0003,MaxForce,Maximiser la production de force: de la molécule au tissu(2014), Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Dynamiques Cellulaire et Tissulaire - Interdisciplinarité, Modèles & Microscopies (TIMC-IMAG-DyCTiM), Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications Grenoble - UMR 5525 (TIMC-IMAG), VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP ), Université Grenoble Alpes (UGA)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP ), Université Grenoble Alpes (UGA), and Aix Marseille Université (AMU)-Collège de France (CdF (institution))-Centre National de la Recherche Scientifique (CNRS)
Subjects: Materials science, Quantitative Biology::Tissues and Organs, Biophysics, macromolecular substances, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Polymerization, Quantitative Biology::Cell Behavior, Protein filament, Focal adhesion, Quantitative Biology::Subcellular Processes, 03 medical and health sciences, 0302 clinical medicine, Rigidity (electromagnetism), Mathematics::Algebraic Geometry, Actin filament dynamics, Cytoskeleton, Actin, 030304 developmental biology, Mechanical Phenomena, 0303 health sciences, biology, Articles, Elasticity, Biomechanical Phenomena, Actin Cytoskeleton, Bundle, Formins, biology.protein, Filopodia, 030217 neurology & neurosurgery, Model
Abstract: International audience; Bundles of actin filaments are central to a large variety of cellular structures such as filopodia, stress fibers, cytokinetic rings, and focal adhesions. The mechanical properties of these bundles are critical for proper force transmission and force bearing. Previous mathematical modeling efforts have focused on bundles' rigidity and shape. However, it remains unknown how bundle length and buckling are controlled by external physical factors. In this work, we present a biophysical model for dynamic bundles of actin filaments submitted to an external load. In combination with in vitro motility assays of beads coated with formins, our model allowed us to characterize conditions for bead movement and bundle buckling. From the deformation profiles, we determined key biophysical properties of tethered actin bundles such as their rigidity and filament density.
Published: 2020
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46. Autoregulatory control of the stability and plasticity of cytoskeletal networks

Author: Ondrej Kucera, Jérémie Gaillard, Christophe Guérin, Manuel Théry, and Laurent Blanchoin
Subjects: Biophysics
Published: 2022
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47. Actin Filament Strain Promotes Severing and Cofilin Dissociation

Author: Laurent Blanchoin, Glen M. Hocky, Gregory A. Voth, Jean Louis Martiel, Anthony C. Schramm, Enrique M. De La Cruz, Department of Molecular Biophysics and Biochemistry, Yale University [New Haven], James Franck Institute, University of Chicago, Physiologie cellulaire et végétale (LPCV), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Dynamique Cellulaire et Tissulaire- Interdisciplinarité, Modèles & Microscopies (TIMC-IMAG-DyCTiM), Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications, Grenoble - UMR 5525 (TIMC-IMAG), Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), National Institutes of Health (NIH) through grant R01-GM097348, Department of Defense Army Research Office through Multidisciplinary University Research Initiative (MURI) grant W911NF1410403, Ruth L. Kirschstein National Research Service Award (National Institute of General Medical Sciences (NIGMS), F32 GM11345-01), ANR-14-CE11-0003,MaxForce,Maximiser la production de force: de la molécule au tissu(2014), Dynamiques Cellulaire et Tissulaire - Interdisciplinarité, Modélisation & Microscopie (TIMC-IMAG-DyCTiM2), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP)-IMAG-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP)-IMAG-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), ANR-14-CE11-0003-01,MaxForce, and Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)
Subjects: 0301 basic medicine, Cofilin severing, Rotation, [SDV]Life Sciences [q-bio], cofilactin, Biophysics, Mechanical properties, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, macromolecular substances, Molecular Dynamics Simulation, MESH: Actins, Quantitative Biology::Cell Behavior, Quantitative Biology::Subcellular Processes, Protein filament, 03 medical and health sciences, Molecular dynamics, MESH: Rotation, MESH: Actin Depolymerizing Factors, Actin filament dynamics, MESH: Protein Binding, Molecular Machines, Motors, and Nanoscale Biophysics, MESH: Molecular Dynamics Simulation, Actin-binding protein, Cytoskeleton, Actin, biology, Chemistry, Computational model, Cryoelectron Microscopy, Elastic energy, Actin binding protein, Cofilin, Actin cytoskeleton, Actins, Elasticity, Actin Cytoskeleton, Crystallography, 030104 developmental biology, Actin Depolymerizing Factors, MESH: Elasticity, biology.protein, MESH: Cryoelectron Microscopy, MESH: Actin Cytoskeleton, Protein Binding
Abstract: International audience; Computational and structural studies have been indispensable in investigating the molecular origins of actin filament mechanical properties and modulation by the regulatory severing protein cofilin. All-atom molecular dynamics simulations of cofilactin filament structures determined by electron cryomicroscopy reveal how cofilin enhances the bending and twisting compliance of actin filaments. Continuum mechanics models suggest that buckled cofilactin filaments localize elastic energy at boundaries between bare and cofilin-decorated segments because of their nonuniform elasticity, thereby accelerating filament severing. Here, we develop mesoscopic length-scale (cofil)actin filament models and evaluate the effects of compressive and twisting loads on strain energy distribution at specific interprotein interfaces. The models reliably capture the filament bending and torsional rigidities and intersubunit torsional flexibility measured experimentally with purified protein components. Buckling is predicted to enhance cofilactin filament severing with minimal effects on cofilin occupancy, whereas filament twisting enhances cofilin dissociation without compromising filament integrity. Preferential severing at actin-cofilactin boundaries of buckled filaments is more prominent than predicted by continuum models because of the enhanced spatial resolution. The models developed here will be valuable for evaluating the effects of filament shape deformations on filament stability and interactions with regulatory proteins, and analysis of single filament manipulation assays.
Published: 2017
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48. Lattice defects induce microtubule self-renewal

Author: Ariane Abrieu, Laura Schaedel, Laurent Blanchoin, Charlotte Aumeier, Manuel Théry, Denis Chrétien, Sarah Triclin, Karin John, Jérémie Gaillard, CytoMorphoLab, Physiologie cellulaire et végétale (LPCV), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Génétique et Développement de Rennes (IGDR), Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )-Centre National de la Recherche Scientifique (CNRS)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES), Centre de recherche en Biologie Cellulaire (CRBM), Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Université Montpellier 1 (UM1), Laboratoire Interdisciplinaire de Physique [Saint Martin d’Hères] (LIPhy), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), French National Research Agency (ANR) ANR-16-CE11-0017-01 ANR-12-BSV5-0004-01 ANR-14-CE09-0014-02 ANR-18-CE13-0001, Human Frontier Science Program RGY0088, European Research Council (ERC) 771599 741773, Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Centre de recherche en Biologie cellulaire de Montpellier (CRBM), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), ANR-18-CE13-0001,APERTuRe,Une nouvelle perspective sur la régulation des microtubules(2018), Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Physiologie cellulaire et végétale (LPCV), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut National de la Recherche Agronomique (INRA), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), and Université Montpellier 1 (UM1)-Université Montpellier 2 - Sciences et Techniques (UM2)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Lattice dynamics, Dimer, [SDV]Life Sciences [q-bio], General Physics and Astronomy, Crystal structure, Self renewal, Instability, 01 natural sciences, Microtubules, Article, Lattice vibrations, 010305 fluids & plasmas, Quantitative Biology::Subcellular Processes, 03 medical and health sciences, chemistry.chemical_compound, Microtubule, Lattice (order), 0103 physical sciences, 010306 general physics, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Physics, Thermal forces, 0303 health sciences, Structural defect, biology, Chemistry, 030302 biochemistry & molecular biology, Model mechanisms, Dissipative dynamics, Dynamics, Tubulin, Lattice defects, biology.protein, Biophysics, Lattice structures, Passive materials, Defects
Abstract: The dynamic instability of microtubules is powered by the addition and removal of tubulin dimers at the ends of the microtubule. Apart from the end, the microtubule shaft is not considered to be dynamic. However recent evidence suggests that free dimers can be incorporated into the shaft of a microtubule damaged by mechanical stress. Here we explored whether dimer exchange was a core property of the microtubule lattice independently of any external constraint. We found that dimers can be removed from and incorporated into the lattice at sites along the microtubule shaft. Furthermore, we showed by experiment and by modeling that rapid dimer renewal requires structural defects in the lattice, which occur in fast growing microtubules. Hence long-lived microtubules have the capacity to self-renew despite their apparent stability and thereby can potentially regulate signaling pathways and structural rearrangements associated with tubulin-dimer exchange at sites along their entire length.
Published: 2019
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49. CLASP mediates microtubule repair by promoting tubulin incorporation into damaged lattices

Author: Laurent Blanchoin, Laura Schaedel, Karin John, Dipti Rai, Amol Aher, Jérémie Gaillard, Manuel Théry, and Anna Akhmanova
Subjects: 0303 health sciences, Microtubule disassembly, biology, Chemistry, Motility, In vitro, 03 medical and health sciences, 0302 clinical medicine, Tubulin, Microtubule, Biophysics, biology.protein, Laser microsurgery, 030217 neurology & neurosurgery, Intracellular, 030304 developmental biology, Microtubule nucleation
Abstract: SummaryMicrotubule network plays a key role in cell division, motility and intracellular trafficking. Microtubule lattices are generally regarded as stable structures that undergo turnover through dynamic instability of their ends [1]. However, recent evidence suggests that microtubules also exchange tubulin dimers at the sites of lattice defects, which can either be induced by mechanical stress or occur spontaneously during polymerization [2–4]. Tubulin incorporation can restore microtubule integrity; moreover, “islands” of freshly incorporated GTP-tubulin can inhibit microtubule disassembly and promote rescues [3–7]. Microtubule repair occurs in vitro in the presence of tubulin alone [2–4, 8]. However, in cells, it is likely to be regulated by specific factors, the nature of which is currently unknown. CLASP is an interesting candidate for microtubule repair, because it induces microtubule nucleation, stimulates rescue and suppresses catastrophes by stabilizing incomplete growing plus ends with lagging protofilaments and promoting their conversion into complete ones [9–16]. Here, we used in vitro reconstitution assays combined with laser microsurgery and microfluidics to show that CLASP2α indeed stimulates microtubule lattice repair. CLASP2α promoted tubulin incorporation into damaged lattice sites thereby restoring microtubule integrity. Furthermore, it induced the formation of complete tubes from partial protofilament assemblies and inhibited microtubule softening caused by hydrodynamic flow-induced bending. A single CLASP2α domain, TOG2, which suppresses catastrophes when tethered to microtubules, was sufficient to stimulate microtubule repair, indicating that catastrophe suppression and lattice repair are mechanistically similar. Our results suggest that the cellular machinery controlling microtubule nucleation and growth can also help to maintain microtubule integrity.
Published: 2019
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50. Spatial integration of mechanical forces by α-actinin establishes actin network symmetry

Author: Laurent Blanchoin, Amandine Pitaval, Hajer Ennomani, Manuel Théry, Laetitia Kurzawa, Fabrice Senger, CytoMorphoLab, Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Physiologie cellulaire et végétale (LPCV), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), ANR-14-CE11-0003-01,MaxForce, European Project: 771599,ICEBERG, European Project: 741773,AAA, Physiologie cellulaire et végétale (LPCV), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), European Research Council (ERC) 741773 771599, French National Research Agency (ANR) ANR-14-CE11-0003-01, and ANR-14-CE11-0003,MaxForce,Maximiser la production de force: de la molécule au tissu(2014)
Subjects: Cell architecture, Retinal Pigment Epithelium, macromolecular substances, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Protein filament, 03 medical and health sciences, Symmetry, 0302 clinical medicine, Crosslinker, Extracellular, Humans, Actinin, Actin, 030304 developmental biology, 0303 health sciences, Network architecture, Crosslinkers, α-Actinin, Cell Biology, Actin cytoskeleton, Symmetry (physics), Actins, Actinin, alpha 1, Mechanical force, Biophysics, Alpha-actinin, Symmery, Mechanical forces, 030217 neurology & neurosurgery, Intracellular
Abstract: Cell and tissue morphogenesis depend on the production and spatial organization of tensional forces in the actin cytoskeleton. Actin network architecture is made of distinct modules characterized by specific filament organizations. The assembly of these modules are well described, but their integration in a cellular network is less understood. Here, we investigated the mechanism regulating the interplay between network architecture and the geometry of the extracellular environment of the cell. We found that α-actinin, a filament crosslinker, is essential for network symmetry to be consistent with extracellular microenvironment symmetry. It is required for the interconnection of transverse arcs with radial fibres to ensure an appropriate balance between forces at cell adhesions and across the actin network. Furthermore, this connectivity appeared necessary for the ability of the cell to integrate and to adapt to complex patterns of extracellular cues as they migrate. Our study has unveiled a role of actin filament crosslinking in the spatial integration of mechanical forces that ensures the adaptation of intracellular symmetry axes in accordance with the geometry of extracellular cues.This article has an associated First Person interview with the first author of the paper.
Published: 2019
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