Motilité structurale

Publications de l’équipe

Année de publication : 2018

Florian Blanc, Tatiana Isabet, Hannah Benisty, H Lee Sweeney, Marco Cecchini, Anne Houdusse (2018 May 31)

An intermediate along the recovery stroke of myosin VI revealed by X-ray crystallography and molecular dynamics.

Proceedings of the National Academy of Sciences of the United States of America : 6213-6218 : DOI : 10.1073/pnas.1711512115 En savoir plus
Résumé

Myosins form a class of actin-based, ATPase motor proteins that mediate important cellular functions such as cargo transport and cell motility. Their functional cycle involves two large-scale swings of the lever arm: the force-generating powerstroke, which takes place on actin, and the recovery stroke during which the lever arm is reprimed into an armed configuration. Previous analyses of the prerecovery (postrigor) and postrecovery (prepowerstroke) states predicted that closure of switch II in the ATP binding site precedes the movement of the converter and the lever arm. Here, we report on a crystal structure of myosin VI, called pretransition state (PTS), which was solved at 2.2 Å resolution. Structural analysis and all-atom molecular dynamics simulations are consistent with PTS being an intermediate along the recovery stroke, where the Relay/SH1 elements adopt a postrecovery conformation, and switch II remains open. In this state, the converter appears to be largely uncoupled from the motor domain and explores an ensemble of partially reprimed configurations through extensive, reversible fluctuations. Moreover, we found that the free energy cost of hydrogen-bonding switch II to ATP is lowered by more than 10 kcal/mol compared with the prerecovery state. These results support the conclusion that closing of switch II does not initiate the recovery stroke transition in myosin VI. Rather, they suggest a mechanism in which lever arm repriming would be mostly driven by thermal fluctuations and eventually stabilized by the switch II interaction with the nucleotide in a ratchet-like fashion.

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Année de publication : 2017

Herschel S Dhekne, Olena Pylypenko, Arend W Overeem, Rosaria J Ferreira, K Joeri van der Velde, Edmond H H M Rings, Carsten Posovszky, Morris A Swertz, Anne Houdusse, Sven C D van IJzendoorn (2017 Dec 22)

MYO5B, STX3 and STXBP2 mutations reveal a common disease mechanism that unifies a subset of congenital diarrheal disorders: A mutation update.

Human mutation : DOI : 10.1002/humu.23386 En savoir plus
Résumé

Microvillus inclusion disease (MVID) is a rare but fatal autosomal recessive congenital diarrheal disorder caused by MYO5B mutations. In 2013 we launched an open-access registry for MVID patients and their MYO5B mutations (www.mvid-central.org). Since then, additional unique MYO5B mutations have been identified in MVID patients, but also in non-MVID patients. Animal models have been generated that formally prove the causality between MYO5B and MVID. Importantly, mutations in two other genes, STXBP2 and STX3, have since been associated with variants of MVID, shedding new light on the pathogenesis of this congenital diarrheal disorder. Here we review these additional genes and their mutations. Furthermore, we discuss recent data from cell studies that indicate that the three genes are functionally linked and, therefore, may constitute a common disease mechanism that unifies a subset of phenotypically linked congenital diarrheal disorders. We present new data based on patient material to support this. To congregate existing and future information on MVID geno-/phenotypes, we have updated and expanded the MVID registry to include all currently known MVID-associated gene mutations, their demonstrated or predicted functional consequences, and associated clinical information. This article is protected by copyright. All rights reserved.

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Sara Bizzotto, Ana Uzquiano, Florent Dingli, Dmitry Ershov, Anne Houllier, Guillaume Arras, Mark Richards, Damarys Loew, Nicolas Minc, Alexandre Croquelois, Anne Houdusse, Fiona Francis (2017 Dec 13)

Eml1 loss impairs apical progenitor spindle length and soma shape in the developing cerebral cortex.

Scientific reports : 17308 : DOI : 10.1038/s41598-017-15253-4 En savoir plus
Résumé

The ventricular zone (VZ) of the developing cerebral cortex is a pseudostratified epithelium that contains progenitors undergoing precisely regulated divisions at its most apical side, the ventricular lining (VL). Mitotic perturbations can contribute to pathological mechanisms leading to cortical malformations. The HeCo mutant mouse exhibits subcortical band heterotopia (SBH), likely to be initiated by progenitor delamination from the VZ early during corticogenesis. The causes for this are however, currently unknown. Eml1, a microtubule (MT)-associated protein of the EMAP family, is impaired in these mice. We first show that MT dynamics are perturbed in mutant progenitor cells in vitro. These may influence interphase and mitotic MT mechanisms and indeed, centrosome and primary cilia were altered and spindles were found to be abnormally long in HeCo progenitors. Consistently, MT and spindle length regulators were identified in EML1 pulldowns from embryonic brain extracts. Finally, we found that mitotic cell shape is also abnormal in the mutant VZ. These previously unidentified VZ characteristics suggest altered cell constraints which may contribute to cell delamination.

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Stéphanie Miserey-Lenkei, Hugo Bousquet, Olena Pylypenko, Sabine Bardin, Ariane Dimitrov, Gaëlle Bressanelli, Raja Bonifay, Vincent Fraisier, Catherine Guillou, Cécile Bougeret, Anne Houdusse, Arnaud Echard, Bruno Goud (2017 Nov 3)

Coupling fission and exit of RAB6 vesicles at Golgi hotspots through kinesin-myosin interactions.

Nature communications : 1254 : DOI : 10.1038/s41467-017-01266-0 En savoir plus
Résumé

The actin and microtubule cytoskeletons play important roles in Golgi structure and function, but how they are connected remain poorly known. In this study, we investigated whether RAB6 GTPase, a Golgi-associated RAB involved in the regulation of several transport steps at the Golgi level, and two of its effectors, Myosin IIA and KIF20A participate in the coupling between actin and microtubule cytoskeleton. We have previously shown that RAB6-Myosin IIA interaction is critical for the fission of RAB6-positive transport carriers from Golgi/TGN membranes. Here we show that KIF20A is also involved in the fission process and serves to anchor RAB6 on Golgi/TGN membranes near microtubule nucleating sites. We provide evidence that the fission events occur at a limited number of hotspots sites. Our results suggest that coupling between actin and microtubule cytoskeletons driven by Myosin II and KIF20A ensures the spatial coordination between RAB6-positive vesicles fission from Golgi/TGN membranes and their exit along microtubules.

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Joseph Atherton, I-Mei Yu, Alexander Cook, Joseph M Muretta, Agnel Joseph, Jennifer Major, Yannick Sourigues, Jeffrey Clause, Maya Topf, Steven S Rosenfeld, Anne Houdusse, Carolyn A Moores (2017 Aug 23)

The divergent mitotic kinesin MKLP2 exhibits atypical structure and mechanochemistry.

eLife : DOI : 10.7554/eLife.27793 En savoir plus
Résumé

MKLP2, a kinesin-6, has critical roles during the metaphase-anaphase transition and cytokinesis. Its motor domain contains conserved nucleotide binding motifs, but is divergent in sequence (~35% identity) and size (~40% larger) compared to other kinesins. Using cryo-electron microscopy and biophysical assays, we have undertaken a mechanochemical dissection of the microtubule-bound MKLP2 motor domain during its ATPase cycle, and show that many facets of its mechanism are distinct from other kinesins. While the MKLP2 neck-linker is directed towards the microtubule plus-end in an ATP-like state, it does not fully dock along the motor domain. Furthermore, the footprint of the MKLP2 motor domain on the MT surface is altered compared to motile kinesins, and enhanced by kinesin-6-specific sequences. The conformation of the highly extended loop6 insertion characteristic of kinesin-6s is nucleotide-independent and does not contact the MT surface. Our results emphasize the role of family-specific insertions in modulating kinesin motor function.

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Vicente J Planelles-Herrero, James J Hartman, Julien Robert-Paganin, Fady I Malik, Anne Houdusse (2017 Aug 5)

Mechanistic and structural basis for activation of cardiac myosin force production by omecamtiv mecarbil.

Nature communications : 190 : DOI : 10.1038/s41467-017-00176-5 En savoir plus
Résumé

Omecamtiv mecarbil is a selective, small-molecule activator of cardiac myosin that is being developed as a potential treatment for heart failure with reduced ejection fraction. Here we determine the crystal structure of cardiac myosin in the pre-powerstroke state, the most relevant state suggested by kinetic studies, both with (2.45 Å) and without (3.10 Å) omecamtiv mecarbil bound. Omecamtiv mecarbil does not change the motor mechanism nor does it influence myosin structure. Instead, omecamtiv mecarbil binds to an allosteric site that stabilizes the lever arm in a primed position resulting in accumulation of cardiac myosin in the primed state prior to onset of cardiac contraction, thus increasing the number of heads that can bind to the actin filament and undergo a powerstroke once the cardiac cycle starts. The mechanism of action of omecamtiv mecarbil also provides insights into uncovering how force is generated by molecular motors.Omecamtiv mecarbil (OM) is a cardiac myosin activator that is currently in clinical trials for heart failure treatment. Here, the authors give insights into its mode of action and present the crystal structure of OM bound to bovine cardiac myosin, which shows that OM stabilizes the pre-powerstroke state of myosin.

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I-Mei Yu, Vicente J Planelles-Herrero, Yannick Sourigues, Dihia Moussaoui, Helena Sirkia, Carlos Kikuti, David Stroebel, Margaret A Titus, Anne Houdusse (2017 Jun 30)

Myosin 7 and its adaptors link cadherins to actin.

Nature communications : 15864 : DOI : 10.1038/ncomms15864 En savoir plus
Résumé

Cadherin linkages between adjacent stereocilia and microvilli are essential for mechanotransduction and maintaining their organization. They are anchored to actin through interaction of their cytoplasmic domains with related tripartite complexes consisting of a class VII myosin and adaptor proteins: Myo7a/SANS/Harmonin in stereocilia and Myo7b/ANKS4B/Harmonin in microvilli. Here, we determine high-resolution structures of Myo7a and Myo7b C-terminal MyTH4-FERM domain (MF2) and unveil how they recognize harmonin using a novel binding mode. Systematic definition of interactions between domains of the tripartite complex elucidates how the complex assembles and prevents possible self-association of harmonin-a. Several Myo7a deafness mutants that map to the surface of MF2 disrupt harmonin binding, revealing the molecular basis for how they impact the formation of the tripartite complex and disrupt mechanotransduction. Our results also suggest how switching between different harmonin isoforms can regulate the formation of networks with Myo7a motors and coordinate force sensing in stereocilia.

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Olena Pylypenko, Hussein Hammich, I-Mei Yu, Anne Houdusse (2017 Jun 21)

Rab GTPases and their interacting protein partners: structural insights into Rab functional diversity.

Small GTPases : 0 : DOI : 10.1080/21541248.2017.1336191 En savoir plus
Résumé

Rab molecular switches are key players in defining membrane identity and regulating intracellular trafficking events in eukaryotic cells. In spite of their global structural similarity, Rab-family members acquired particular features that allow them to perform specific cellular functions. The overall fold and local sequence conservations enable them to utilize a common machinery for prenylation and recycling; while individual Rab structural differences determine interactions with specific partners such as GEFs, GAPs and effector proteins. These interactions orchestrate the spatiotemporal regulation of Rab localization and their turning ON and OFF, leading to tightly controlled Rab-specific functionalities such as membrane composition modifications, recruitment of molecular motors for intracellular trafficking, or recruitment of scaffold proteins that mediate interactions with downstream partners, as well as actin cytoskeleton regulation. In this review we summarize structural information on Rab GTPases and their complexes with protein partners in the context of partner binding specificity and functional outcomes of their interactions in the cell.

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Stéphane Frémont, Guillaume Romet-Lemonne, Anne Houdusse, Arnaud Echard (2017 Apr 5)

Emerging roles of MICAL family proteins – from actin oxidation to membrane trafficking during cytokinesis.

Journal of cell science : 1509-1517 : DOI : 10.1242/jcs.202028 En savoir plus
Résumé

Cytokinetic abscission is the terminal step of cell division, leading to the physical separation of the two daughter cells. The exact mechanism mediating the final scission of the intercellular bridge connecting the dividing cells is not fully understood, but requires the local constriction of endosomal sorting complex required for transport (ESCRT)-III-dependent helices, as well as remodelling of lipids and the cytoskeleton at the site of abscission. In particular, microtubules and actin filaments must be locally disassembled for successful abscission. However, the mechanism that actively removes actin during abscission is poorly understood. In this Commentary, we will focus on the latest findings regarding the emerging role of the MICAL family of oxidoreductases in F-actin disassembly and describe how Rab GTPases regulate their enzymatic activity. We will also discuss the recently reported role of MICAL1 in controlling F-actin clearance in the ESCRT-III-mediated step of cytokinetic abscission. In addition, we will highlight how two other members of the MICAL family (MICAL3 and MICAL-L1) contribute to cytokinesis by regulating membrane trafficking. Taken together, these findings establish the MICAL family as a key regulator of actin cytoskeleton dynamics and membrane trafficking during cell division.

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Stéphane Frémont, Hussein Hammich, Jian Bai, Hugo Wioland, Kerstin Klinkert, Murielle Rocancourt, Carlos Kikuti, David Stroebel, Guillaume Romet-Lemonne, Olena Pylypenko, Anne Houdusse, Arnaud Echard (2017 Feb 24)

Oxidation of F-actin controls the terminal steps of cytokinesis.

Nature communications : 14528 : DOI : 10.1038/ncomms14528 En savoir plus
Résumé

Cytokinetic abscission, the terminal step of cell division, crucially depends on the local constriction of ESCRT-III helices after cytoskeleton disassembly. While the microtubules of the intercellular bridge are cut by the ESCRT-associated enzyme Spastin, the mechanism that clears F-actin at the abscission site is unknown. Here we show that oxidation-mediated depolymerization of actin by the redox enzyme MICAL1 is key for ESCRT-III recruitment and successful abscission. MICAL1 is recruited to the abscission site by the Rab35 GTPase through a direct interaction with a flat three-helix domain found in MICAL1 C terminus. Mechanistically, in vitro assays on single actin filaments demonstrate that MICAL1 is activated by Rab35. Moreover, in our experimental conditions, MICAL1 does not act as a severing enzyme, as initially thought, but instead induces F-actin depolymerization from both ends. Our work reveals an unexpected role for oxidoreduction in triggering local actin depolymerization to control a fundamental step of cell division.

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Année de publication : 2016

Karl J Petersen, Holly V Goodson, Ashley L Arthur, G W Gant Luxton, Anne Houdusse, Margaret A Titus (2016 Dec 3)

MyTH4-FERM myosins have an ancient and conserved role in filopod formation.

Proceedings of the National Academy of Sciences of the United States of America : DOI : 201615392 En savoir plus
Résumé

The formation of filopodia in Metazoa and Amoebozoa requires the activity of myosin 10 (Myo10) in mammalian cells and of Dictyostelium unconventional myosin 7 (DdMyo7) in the social amoeba Dictyostelium However, the exact roles of these MyTH4-FERM myosins (myosin tail homology 4-band 4.1, ezrin, radixin, moesin; MF) in the initiation and elongation of filopodia are not well defined and may reflect conserved functions among phylogenetically diverse MF myosins. Phylogenetic analysis of MF myosin domains suggests that a single ancestral MF myosin existed with a structure similar to DdMyo7, which has two MF domains, and that subsequent duplications in the metazoan lineage produced its functional homolog Myo10. The essential functional features of the DdMyo7 myosin were identified using quantitative live-cell imaging to characterize the ability of various mutants to rescue filopod formation in myo7-null cells. The two MF domains were found to function redundantly in filopod formation with the C-terminal FERM domain regulating both the number of filopodia and their elongation velocity. DdMyo7 mutants consisting solely of the motor plus a single MyTH4 domain were found to be capable of rescuing the formation of filopodia, establishing the minimal elements necessary for the function of this myosin. Interestingly, a chimeric myosin with the Myo10 MF domain fused to the DdMyo7 motor also was capable of rescuing filopod formation in the myo7-null mutant, supporting fundamental functional conservation between these two distant myosins. Together, these findings reveal that MF myosins have an ancient and conserved role in filopod formation.

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Sirigu S, Hartman J, Planelles-Herrero VJ, Ropars V, Clancy S, Wang X, Chuang G, Qian X, Lu P-P, Barrett E, Rudolph K, Royer C, Morgan B, Stura EA, Malik FI, Houdusse A (2016 Nov 4)

Highly selective inhibition of myosin motors provides the basis of potential therapeutic application.

Proceedings of the National Academy of Sciences of the United States of America : 201609342 : DOI : 10.1073/pnas.1609342113 En savoir plus
Résumé

Direct inhibition of smooth muscle myosin (SMM) is a potential means to treat hypercontractile smooth muscle diseases. The selective inhibitor CK-2018571 prevents strong binding to actin and promotes muscle relaxation in vitro and in vivo. The crystal structure of the SMM/drug complex reveals that CK-2018571 binds to a novel allosteric pocket that opens up during the « recovery stroke » transition necessary to reprime the motor. Trapped in an intermediate of this fast transition, SMM is inhibited with high selectivity compared with skeletal muscle myosin (IC50 = 9 nM and 11,300 nM, respectively), although all of the binding site residues are identical in these motors. This structure provides a starting point from which to design highly specific myosin modulators to treat several human diseases. It further illustrates the potential of targeting transition intermediates of molecular machines to develop exquisitely selective pharmacological agents.

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Anne Houdusse, H Lee Sweeney (2016 Oct 9)

How Myosin Generates Force on Actin Filaments.

Trends in biochemical sciences : DOI : S0968-0004(16)30152-9 En savoir plus
Résumé

How myosin interacts with actin to generate force is a subject of considerable controversy. The major debate centers on understanding at what point in force generation the inorganic phosphate is released with respect to the lever arm swing, or powerstroke. Resolving the controversy is essential for understanding how force is produced as well as the mechanisms underlying disease-causing mutations in myosin. Recent structural insights into the powerstroke have come from a high-resolution structure of myosin in a previously unseen state and from an electron cryomicroscopy (cryo-EM) 3D reconstruction of the actin-myosin-MgADP complex. Here, we argue that seemingly contradictory data from time-resolved fluorescence resonance energy transfer (FRET) studies can be reconciled, and we put forward a model for myosin force generation on actin.

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Pylypenko O, Welz T, Tittel J, Kollmar M, Chardon F, Malherbe G, Weiss S, Michel C, Samol-Wolf A, Grasskamp A, Hume A, Goud B, Baron B, England P, Titus MA, Schwille P, Weidemann T, Houdusse A, Kerkhoff E (2016 Sep 14)

Coordinated recruitment of Spir actin nucleators and myosin V motors to Rab11 vesicle membranes

eLife : 5 : e17523 : DOI : 10.7554/eLife.17523 En savoir plus
Résumé

There is growing evidence for a coupling of actin assembly and myosin motor activity in cells. However, mechanisms for recruitment of actin nucleators and motors on specific membrane compartments remain unclear. Here we report how Spir actin nucleators and myosin V motors coordinate their specific membrane recruitment. The myosin V globular tail domain (MyoV-GTD) interacts directly with an evolutionarily conserved Spir sequence motif. We determined crystal structures of MyoVa-GTD bound either to the Spir-2 motif or to Rab11 and show that a Spir-2:MyoVa:Rab11 complex can form. The ternary complex architecture explains how Rab11 vesicles support coordinated F-actin nucleation and myosin force generation for vesicle transport and tethering. New insights are also provided into how myosin activation can be coupled with the generation of actin tracks. Since MyoV binds several Rab GTPases, synchronized nucleator and motor targeting could provide a common mechanism to control force generation and motility in different cellular processes.

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Virginie Ropars, Zhaohui Yang, Tatiana Isabet, Florian Blanc, Kaifeng Zhou, Tianming Lin, Xiaoyan Liu, Pascale Hissier, Frédéric Samazan, Béatrice Amigues, Eric D Yang, Hyokeun Park, Olena Pylypenko, Marco Cecchini, Charles V Sindelar, H Lee Sweeney, Anne Houdusse (2016 Sep 2)

The myosin X motor is optimized for movement on actin bundles.

Nature communications : 12456 : DOI : 10.1038/ncomms12456 En savoir plus
Résumé

Myosin X has features not found in other myosins. Its structure must underlie its unique ability to generate filopodia, which are essential for neuritogenesis, wound healing, cancer metastasis and some pathogenic infections. By determining high-resolution structures of key components of this motor, and characterizing the in vitro behaviour of the native dimer, we identify the features that explain the myosin X dimer behaviour. Single-molecule studies demonstrate that a native myosin X dimer moves on actin bundles with higher velocities and takes larger steps than on single actin filaments. The largest steps on actin bundles are larger than previously reported for artificially dimerized myosin X constructs or any other myosin. Our model and kinetic data explain why these large steps and high velocities can only occur on bundled filaments. Thus, myosin X functions as an antiparallel dimer in cells with a unique geometry optimized for movement on actin bundles.

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