Motilité structurale

Publications de l’équipe

Année de publication : 2019

Julien Robert-Paganin, Olena Pylypenko, Carlos Kikuti, H Lee Sweeney, Anne Houdusse (2019 Nov 6)

Force Generation by Myosin Motors: A Structural Perspective.

Chemical reviews : 5-35 : DOI : 10.1021/acs.chemrev.9b00264 En savoir plus
Résumé

Generating force and movement is essential for the functions of cells and organisms. A variety of molecular motors that can move on tracks within cells have evolved to serve this role. How these motors interact with their tracks and how that, in turn, leads to the generation of force and movement is key to understanding the cellular roles that these motor-track systems serve. This review is focused on the best understood of these systems, which is the molecular motor myosin that moves on tracks of filamentous (F-) actin. The review highlights both the progress and the limits of our current understanding of how force generation can be controlled by F-actin-myosin interactions. What has emerged are insights they may serve as a framework for understanding the design principles of a number of types of molecular motors and their interactions with their tracks.

Replier
Ashley L Arthur, Livia D Songster, Helena Sirkia, Akash Bhattacharya, Carlos Kikuti, Fernanda Pires Borrega, Anne Houdusse, Margaret A Titus (2019 Oct 16)

Optimized filopodia formation requires myosin tail domain cooperation.

Proceedings of the National Academy of Sciences of the United States of America : 22196-22204 : DOI : 10.1073/pnas.1901527116 En savoir plus
Résumé

Filopodia are actin-filled protrusions employed by cells to interact with their environment. Filopodia formation in Amoebozoa and Metazoa requires the phylogenetically diverse MyTH4-FERM (MF) myosins DdMyo7 and Myo10, respectively. While Myo10 is known to form antiparallel dimers, DdMyo7 lacks a coiled-coil domain in its proximal tail region, raising the question of how such divergent motors perform the same function. Here, it is shown that the DdMyo7 lever arm plays a role in both autoinhibition and function while the proximal tail region can mediate weak dimerization, and is proposed to be working in cooperation with the C-terminal MF domain to promote partner-mediated dimerization. Additionally, a forced dimer of the DdMyo7 motor is found to weakly rescue filopodia formation, further highlighting the importance of the C-terminal MF domain. Thus, weak dimerization activity of the DdMyo7 proximal tail allows for sensitive regulation of myosin activity to prevent inappropriate activation of filopodia formation. The results reveal that the principles of MF myosin-based filopodia formation are conserved via divergent mechanisms for dimerization.

Replier
Julien Robert-Paganin, James P Robblee, Daniel Auguin, Thomas C A Blake, Carol S Bookwalter, Elena B Krementsova, Dihia Moussaoui, Michael J Previs, Guillaume Jousset, Jake Baum, Kathleen M Trybus, Anne Houdusse (2019 Jul 25)

Plasmodium myosin A drives parasite invasion by an atypical force generating mechanism.

Nature communications : 3286 : DOI : 10.1038/s41467-019-11120-0 En savoir plus
Résumé

Plasmodium parasites are obligate intracellular protozoa and causative agents of malaria, responsible for half a million deaths each year. The lifecycle progression of the parasite is reliant on cell motility, a process driven by myosin A, an unconventional single-headed class XIV molecular motor. Here we demonstrate that myosin A from Plasmodium falciparum (PfMyoA) is critical for red blood cell invasion. Further, using a combination of X-ray crystallography, kinetics, and in vitro motility assays, we elucidate the non-canonical interactions that drive this motor’s function. We show that PfMyoA motor properties are tuned by heavy chain phosphorylation (Ser19), with unphosphorylated PfMyoA exhibiting enhanced ensemble force generation at the expense of speed. Regulated phosphorylation may therefore optimize PfMyoA for enhanced force generation during parasite invasion or for fast motility during dissemination. The three PfMyoA crystallographic structures presented here provide a blueprint for discovery of specific inhibitors designed to prevent parasite infection.

Replier
Marco Lucchino, Anne Billet, Antoine Versini, Harikrishna Bavireddi, Bhanu-Das Dasari, Sylvain Debieu, Ludovic Colombeau, Tatiana Cañeque, Alain Wagner, Géraldine Masson, Frédéric Taran, Philippe Karoyan, Muriel Delepierre, Christine Gaillet, Anne Houdusse, Sébastien Britton, Frédéric Schmidt, Jean-Claude Florent, Philippe Belmont, David Monchaud, Janine Cossy, Christophe Thomas, Arnaud Gautier, Ludger Johannes, Raphaël Rodriguez (2019 Feb 26)

2nd PSL Chemical Biology Symposium (2019): At the Crossroads of Chemistry and Biology.

Chembiochem : a European journal of chemical biology : 968-973 : DOI : 10.1002/cbic.201900092 En savoir plus
Résumé

Chemical Biology is the science of designing chemical tools to dissect and manipulate biology at different scales. It provides the fertile ground from which to address important problems of our society, such as human health and environment.

Replier

Année de publication : 2018

Julien Robert-Paganin, Daniel Auguin, Anne Houdusse (2018 Oct 3)

Hypertrophic cardiomyopathy disease results from disparate impairments of cardiac myosin function and auto-inhibition.

Nature communications : 4019 : DOI : 10.1038/s41467-018-06191-4 En savoir plus
Résumé

Hypertrophic cardiomyopathies (HCM) result from distinct single-point mutations in sarcomeric proteins that lead to muscle hypercontractility. While different models account for a pathological increase in the power output, clear understanding of the molecular basis of dysfunction in HCM is the mandatory next step to improve current treatments. Here, we present an optimized quasi-atomic model of the sequestered state of cardiac myosin coupled to X-ray crystallography and in silico analysis of the mechanical compliance of the lever arm, allowing the systematic study of a large set of HCM mutations and the definition of different mutation classes based on their effects on lever arm compliance, sequestered state stability, and motor functions. The present work reconciles previous models and explains how distinct HCM mutations can have disparate effects on the motor mechano-chemical parameters and yet lead to the same disease. The framework presented here can guide future investigations aiming at finding HCM treatments.

Replier
Alison G Tebo, Frederico M Pimenta, Martha Zoumpoulaki, Carlos Kikuti, Helena Sirkia, Marie-Aude Plamont, Anne Houdusse, Arnaud Gautier (2018 Aug 9)

Circularly Permuted Fluorogenic Proteins for the Design of Modular Biosensors.

ACS chemical biology : DOI : 10.1021/acschembio.8b00417 En savoir plus
Résumé

Fluorescent reporters are essential components for the design of optical biosensors that are able to image intracellular analytes in living cells. Herein, we describe the development of circularly permuted variants of Fluorescence-Activating and absorption-Shifting Tag (FAST) and demonstrate their potential as reporting module in biosensors. Circularly permutated FAST (cpFAST) variants allow one to condition the binding and activation of a fluorogenic ligand (and thus fluorescence) to analyte recognition by coupling them with analyte-binding domains. We demonstrated their use for biosensor design by generating multicolor plug-and-play fluorogenic biosensors for imaging the intracellular levels of Ca in living mammalian cells in real time.

Replier
Léa Ripoll, Xavier Heiligenstein, Ilse Hurbain, Lia Domingues, Florent Figon, Karl J Petersen, Megan K Dennis, Anne Houdusse, Michael S Marks, Graça Raposo, Cédric Delevoye (2018 Jun 8)

Myosin VI and branched actin filaments mediate membrane constriction and fission of melanosomal tubule carriers.

The Journal of cell biology : 2709-2726 : DOI : 10.1083/jcb.201709055 En savoir plus
Résumé

Vesicular and tubular transport intermediates regulate organellar cargo dynamics. Transport carrier release involves local and profound membrane remodeling before fission. Pinching the neck of a budding tubule or vesicle requires mechanical forces, likely exerted by the action of molecular motors on the cytoskeleton. Here, we show that myosin VI, together with branched actin filaments, constricts the membrane of tubular carriers that are then released from melanosomes, the pigment containing lysosome-related organelles of melanocytes. By combining superresolution fluorescence microscopy, correlative light and electron microscopy, and biochemical analyses, we find that myosin VI motor activity mediates severing by constricting the neck of the tubule at specific melanosomal subdomains. Pinching of the tubules involves the cooperation of the myosin adaptor optineurin and the activity of actin nucleation machineries, including the WASH and Arp2/3 complexes. The fission and release of these tubules allows for the export of components from melanosomes, such as the SNARE VAMP7, and promotes melanosome maturation and transfer to keratinocytes. Our data reveal a new myosin VI- and actin-dependent membrane fission mechanism required for organelle function.

Replier
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.

Replier

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.

Replier
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.

Replier
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.

Replier
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.

Replier
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.

Replier
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.

Replier
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.

Replier