Motilité structurale

Publications de l’équipe

Année de publication : 2016

Vicente José Planelles-Herrero, Florian Blanc, Serena Sirigu, Helena Sirkia, Jeffrey Clause, Yannick Sourigues, Daniel O Johnsrud, Beatrice Amigues, Marco Cecchini, Susan P Gilbert, Anne Houdusse, Margaret A Titus (2016 May 12)

Myosin MyTH4-FERM structures highlight important principles of convergent evolution.

Proceedings of the National Academy of Sciences of the United States of America : E2906-15 : DOI : 10.1073/pnas.1600736113 En savoir plus
Résumé

Myosins containing MyTH4-FERM (myosin tail homology 4-band 4.1, ezrin, radixin, moesin, or MF) domains in their tails are found in a wide range of phylogenetically divergent organisms, such as humans and the social amoeba Dictyostelium (Dd). Interestingly, evolutionarily distant MF myosins have similar roles in the extension of actin-filled membrane protrusions such as filopodia and bind to microtubules (MT), suggesting that the core functions of these MF myosins have been highly conserved over evolution. The structures of two DdMyo7 signature MF domains have been determined and comparison with mammalian MF structures reveals that characteristic features of MF domains are conserved. However, across millions of years of evolution conserved class-specific insertions are seen to alter the surfaces and the orientation of subdomains with respect to each other, likely resulting in new sites for binding partners. The MyTH4 domains of Myo10 and DdMyo7 bind to MT with micromolar affinity but, surprisingly, their MT binding sites are on opposite surfaces of the MyTH4 domain. The structural analysis in combination with comparison of diverse MF myosin sequences provides evidence that myosin tail domain features can be maintained without strict conservation of motifs. The results illustrate how tuning of existing features can give rise to new structures while preserving the general properties necessary for myosin tails. Thus, tinkering with the MF domain enables it to serve as a multifunctional platform for cooperative recruitment of various partners, allowing common properties such as autoinhibition of the motor and microtubule binding to arise through convergent evolution.

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Kerstin Klinkert, Murielle Rocancourt, Anne Houdusse, Arnaud Echard (2016 Apr 5)

Rab35 GTPase couples cell division with initiation of epithelial apico-basal polarity and lumen opening.

Nature communications : 11166 : DOI : 10.1038/ncomms11166 En savoir plus
Résumé

Establishment and maintenance of apico-basal polarity in epithelial organs must be tightly coupled with cell division, but the underlying molecular mechanisms are largely unknown. Using 3D cultures of renal MDCK cells (cysts), we found that the Rab35 GTPase plays a crucial role in polarity initiation and apical lumen positioning during the first cell division of cyst development. At the molecular level, Rab35 physically couples cytokinesis with the initiation of apico-basal polarity by tethering intracellular vesicles containing key apical determinants at the cleavage site. These vesicles transport aPKC, Cdc42, Crumbs3 and the lumen-promoting factor Podocalyxin, and are tethered through a direct interaction between Rab35 and the cytoplasmic tail of Podocalyxin. Consequently, Rab35 inactivation leads to complete inversion of apico-basal polarity in 3D cysts. This novel and unconventional mode of Rab-dependent vesicle targeting provides a simple mechanism for triggering both initiation of apico-basal polarity and lumen opening at the centre of cysts.

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Sarah F Wulf, Virginie Ropars, Setsuko Fujita-Becker, Marco Oster, Goetz Hofhaus, Leonardo G Trabuco, Olena Pylypenko, H Lee Sweeney, Anne M Houdusse, Rasmus R Schröder (2016 Mar 16)

Force-producing ADP state of myosin bound to actin.

Proceedings of the National Academy of Sciences of the United States of America : E1844-52 : DOI : 10.1073/pnas.1516598113 En savoir plus
Résumé

Molecular motors produce force when they interact with their cellular tracks. For myosin motors, the primary force-generating state has MgADP tightly bound, whereas myosin is strongly bound to actin. We have generated an 8-Å cryoEM reconstruction of this state for myosin V and used molecular dynamics flexed fitting for model building. We compare this state to the subsequent state on actin (Rigor). The ADP-bound structure reveals that the actin-binding cleft is closed, even though MgADP is tightly bound. This state is accomplished by a previously unseen conformation of the β-sheet underlying the nucleotide pocket. The transition from the force-generating ADP state to Rigor requires a 9.5° rotation of the myosin lever arm, coupled to a β-sheet rearrangement. Thus, the structure reveals the detailed rearrangements underlying myosin force generation as well as the basis of strain-dependent ADP release that is essential for processive myosins, such as myosin V.

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Année de publication : 2015

Frédéric Samazan, Bachra Rokbi, Delphine Seguin, Fabienne Telles, Valérie Gautier, Gilbert Richarme, Didier Chevret, Paloma Fernández Varela, Christophe Velours, Isabelle Poquet (2015 Jul 17)

Production, secretion and purification of a correctly folded staphylococcal antigen in Lactococcus lactis.

Microbial cell factories : 104 : DOI : 10.1186/s12934-015-0271-z En savoir plus
Résumé

Lactococcus lactis, a lactic acid bacterium traditionally used to ferment milk and manufacture cheeses, is also, in the biotechnology field, an interesting host to produce proteins of medical interest, as it is « Generally Recognized As Safe ». Furthermore, as L. lactis naturally secretes only one major endogenous protein (Usp45), the secretion of heterologous proteins in this species facilitates their purification from a protein-poor culture medium. Here, we developed and optimized protein production and secretion in L. lactis to obtain proteins of high quality, both correctly folded and pure to a high extent. As proteins to be produced, we chose the two transmembrane members of the HtrA protease family in Staphylococcus aureus, an important extra-cellular pathogen, as these putative surface-exposed antigens could constitute good targets for vaccine development.

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Paola Llinas, Tatiana Isabet, Lin Song, Virginie Ropars, Bin Zong, Hannah Benisty, Serena Sirigu, Carl Morris, Carlos Kikuti, Dan Safer, H Lee Sweeney, Anne Houdusse (2015 May 5)

How actin initiates the motor activity of Myosin.

Developmental cell : 401-12 : DOI : 10.1016/j.devcel.2015.03.025 En savoir plus
Résumé

Fundamental to cellular processes are directional movements driven by molecular motors. A common theme for these and other molecular machines driven by ATP is that controlled release of hydrolysis products is essential for using the chemical energy efficiently. Mechanochemical transduction by myosin motors on actin is coupled to unknown structural changes that result in the sequential release of inorganic phosphate (Pi) and MgADP. We present here a myosin structure possessing an actin-binding interface and a tunnel (back door) that creates an escape route for Pi with a minimal rotation of the myosin lever arm that drives movements. We propose that this state represents the beginning of the powerstroke on actin and that Pi translocation from the nucleotide pocket triggered by actin binding initiates myosin force generation. This elucidates how actin initiates force generation and movement and may represent a strategy common to many molecular machines.

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Olena Pylypenko, Lin Song, Ai Shima, Zhaohui Yang, Anne M Houdusse, H Lee Sweeney (2015 Mar 10)

Myosin VI deafness mutation prevents the initiation of processive runs on actin.

Proceedings of the National Academy of Sciences of the United States of America : E1201-9 : DOI : 10.1073/pnas.1420989112 En savoir plus
Résumé

Mutations in the reverse-direction myosin, myosin VI, are associated with deafness in humans and mice. A myosin VI deafness mutation, D179Y, which is in the transducer of the motor, uncoupled the release of the ATP hydrolysis product, inorganic phosphate (Pi), from dependency on actin binding and destroyed the ability of single dimeric molecules to move processively on actin filaments. We observed that processive movement is rescued if ATP is added to the mutant dimer following binding of both heads to actin in the absence of ATP, demonstrating that the mutation selectively destroys the initiation of processive runs at physiological ATP levels. A drug (omecamtiv) that accelerates the actin-activated activity of cardiac myosin was able to rescue processivity of the D179Y mutant dimers at physiological ATP concentrations by slowing the actin-independent release of Pi. Thus, it may be possible to create myosin VI-specific drugs that rescue the function of deafness-causing mutations.

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Année de publication : 2014

Joseph Atherton, Irene Farabella, I-Mei Yu, Steven S Rosenfeld, Anne Houdusse, Maya Topf, Carolyn A Moores (2014 Sep 12)

Conserved mechanisms of microtubule-stimulated ADP release, ATP binding, and force generation in transport kinesins.

eLife : e03680 : DOI : 10.7554/eLife.03680 En savoir plus
Résumé

Kinesins are a superfamily of microtubule-based ATP-powered motors, important for multiple, essential cellular functions. How microtubule binding stimulates their ATPase and controls force generation is not understood. To address this fundamental question, we visualized microtubule-bound kinesin-1 and kinesin-3 motor domains at multiple steps in their ATPase cycles–including their nucleotide-free states–at ∼ 7 Å resolution using cryo-electron microscopy. In both motors, microtubule binding promotes ordered conformations of conserved loops that stimulate ADP release, enhance microtubule affinity and prime the catalytic site for ATP binding. ATP binding causes only small shifts of these nucleotide-coordinating loops but induces large conformational changes elsewhere that allow force generation and neck linker docking towards the microtubule plus end. Family-specific differences across the kinesin-microtubule interface account for the distinctive properties of each motor. Our data thus provide evidence for a conserved ATP-driven mechanism for kinesins and reveal the critical mechanistic contribution of the microtubule interface.

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Monalisa Mukherjea, M Yusuf Ali, Carlos Kikuti, Daniel Safer, Zhaohui Yang, Helena Sirkia, Virginie Ropars, Anne Houdusse, David M Warshaw, H Lee Sweeney (2014 Aug 28)

Myosin VI must dimerize and deploy its unusual lever arm in order to perform its cellular roles.

Cell reports : 1522-32 : DOI : 10.1016/j.celrep.2014.07.041 En savoir plus
Résumé

It is unclear whether the reverse-direction myosin (myosin VI) functions as a monomer or dimer in cells and how it generates large movements on actin. We deleted a stable, single-α-helix (SAH) domain that has been proposed to function as part of a lever arm to amplify movements without impact on in vitro movement or in vivo functions. A myosin VI construct that used this SAH domain as part of its lever arm was able to take large steps in vitro but did not rescue in vivo functions. It was necessary for myosin VI to internally dimerize, triggering unfolding of a three-helix bundle and calmodulin binding in order to step normally in vitro and rescue endocytosis and Golgi morphology in myosin VI-null fibroblasts. A model for myosin VI emerges in which cargo binding triggers dimerization and unfolds the three-helix bundle to create a lever arm essential for in vivo functions.

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Zippora Brownstein, Amal Abu-Rayyan, Daphne Karfunkel-Doron, Serena Sirigu, Bella Davidov, Mordechai Shohat, Moshe Frydman, Anne Houdusse, Moien Kanaan, Karen B Avraham (2014 Jun 1)

Novel myosin mutations for hereditary hearing loss revealed by targeted genomic capture and massively parallel sequencing.

European journal of human genetics : EJHG : 22 : 768-75 : DOI : 10.1038/ejhg.2013.232 En savoir plus
Résumé

Hereditary hearing loss is genetically heterogeneous, with a large number of genes and mutations contributing to this sensory, often monogenic, disease. This number, as well as large size, precludes comprehensive genetic diagnosis of all known deafness genes. A combination of targeted genomic capture and massively parallel sequencing (MPS), also referred to as next-generation sequencing, was applied to determine the deafness-causing genes in hearing-impaired individuals from Israeli Jewish and Palestinian Arab families. Among the mutations detected, we identified nine novel mutations in the genes encoding myosin VI, myosin VIIA and myosin XVA, doubling the number of myosin mutations in the Middle East. Myosin VI mutations were identified in this population for the first time. Modeling of the mutations provided predicted mechanisms for the damage they inflict in the molecular motors, leading to impaired function and thus deafness. The myosin mutations span all regions of these molecular motors, leading to a wide range of hearing phenotypes, reinforcing the key role of this family of proteins in auditory function. This study demonstrates that multiple mutations responsible for hearing loss can be identified in a relatively straightforward manner by targeted-gene MPS technology and concludes that this is the optimal genetic diagnostic approach for identification of mutations responsible for hearing loss.

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Année de publication : 2013

Olena Pylypenko, Wikayatou Attanda, Charles Gauquelin, Marion Lahmani, Doudouh Coulibaly, Bruno Baron, Sylviane Hoos, Margaret A Titus, Patrick England, Anne M Houdusse (2013 Nov 18)

Structural basis of myosin V Rab GTPase-dependent cargo recognition.

Proceedings of the National Academy of Sciences of the United States of America : 20443-8 : DOI : 10.1073/pnas.1314329110 En savoir plus
Résumé

Specific recognition of the cargo that molecular motors transport or tether to cytoskeleton tracks allows them to perform precise cellular functions at particular times and positions in cells. However, very little is known about how evolution has favored conservation of functions for some isoforms, while also allowing for the generation of new recognition sites and specialized cellular functions. Here we present several crystal structures of the myosin Va or the myosin Vb globular tail domain (GTD) that gives insights into how the motor is linked to the recycling membrane compartments via Rab11 or to the melanosome membrane via recognition of the melanophilin adaptor that binds to Rab27a. The structures illustrate how the Rab11-binding site has been conserved during evolution and how divergence at another site of the GTD allows more specific interactions such as the specific recognition of melanophilin by the myosin Va isoform. With atomic structural insights, these structures also show how either the partner or the GTD structural plasticity upon association is critical for selective recruitment of the motor.

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K Joeri van der Velde, Herschel S Dhekne, Morris A Swertz, Serena Sirigu, Virginie Ropars, Petra C Vinke, Trebor Rengaw, Peter C van den Akker, Edmond H H M Rings, Anne Houdusse, Sven C D van Ijzendoorn (2013 Sep 10)

An overview and online registry of microvillus inclusion disease patients and their MYO5B mutations.

Human mutation : 1597-605 : DOI : 10.1002/humu.22440 En savoir plus
Résumé

Microvillus inclusion disease (MVID) is one of the most severe congenital intestinal disorders and is characterized by neonatal secretory diarrhea and the inability to absorb nutrients from the intestinal lumen. MVID is associated with patient-, family-, and ancestry-unique mutations in the MYO5B gene, encoding the actin-based motor protein myosin Vb. Here, we review the MYO5B gene and all currently known MYO5B mutations and for the first time methodologically categorize these with regard to functional protein domains and recurrence in MYO7A associated with Usher syndrome and other myosins. We also review animal models for MVID and the latest data on functional studies related to the myosin Vb protein. To congregate existing and future information on MVID geno-/phenotypes and facilitate its quick and easy sharing among clinicians and researchers, we have constructed an online MOLGENIS-based international patient registry (www.MVID-central.org). This easily accessible database currently contains detailed information of 137 MVID patients together with reported clinical/phenotypic details and 41 unique MYO5B mutations, of which several unpublished. The future expansion and prospective nature of this registry is expected to improve disease diagnosis, prognosis, and genetic counseling.

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Franck Fourniol, Mylène Perderiset, Anne Houdusse, Carolyn Moores (2013 Aug 27)

Structural studies of the doublecortin family of MAPs.

Methods in cell biology : 27-48 : DOI : 10.1016/B978-0-12-407757-7.00003-7 En savoir plus
Résumé

Doublecortin (DCX) is a microtubule (MT)-stabilizing protein essential for neuronal migration during human brain development. Missense mutations in DCX cause severe brain defects. This implies that the many other MT-stabilizing proteins in neurons cannot compensate for DCX function. To understand the unusual properties of DCX, we expressed the recombinant human DCX in Sf9 cells and undertook structural characterization of its interaction with MTs using cryo-electron microscopy. DCX specifically nucleates 13-protofilament (13-pf) MTs, the architecture of human MTs in vivo. Cryo-electron tomography (cryo-ET) of DCX-nucleated MTs showed that they are primarily built from B-lattice contacts interrupted by a single discontinuity, the seam. Because of this asymmetry, we used single-particle reconstruction and determined the 8Å structure of DCX-stabilized 13-pf MTs in the absence of a stabilizing drug. The DCX-binding site, at the corner of four tubulin dimers, is ideally suited to stabilize both lateral and longitudinal tubulin lattice contacts. Its precise geometry suggests that DCX is sensitive to the angle between pfs, and thereby provides insight into the specificity of DCX for 13-pf MT architecture. DCX’s precise interaction at the corner of four tubulin dimers also means that DCX does not bind the MT seam. Our work has provided mechanistic insight into the evolutionarily conserved DCX family of MT-stabilizing proteins and also into more general regulatory mechanisms of the MT cytoskeleton.

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Joseph Atherton, Anne Houdusse, Carolyn Moores (2013 Jun 26)

MAPping out distribution routes for kinesin couriers.

Biology of the cell / under the auspices of the European Cell Biology Organization : 465-87 : DOI : 10.1111/boc.201300012 En savoir plus
Résumé

In the crowded environment of eukaryotic cells, diffusion is an inefficient distribution mechanism for cellular components. Long-distance active transport is required and is performed by molecular motors including kinesins. Furthermore, in highly polarised, compartmentalised and plastic cells such as neurons, regulatory mechanisms are required to ensure appropriate spatio-temporal delivery of neuronal components. The kinesin machinery has diversified into a large number of kinesin motor proteins as well as adaptor proteins that are associated with subsets of cargo. However, many mechanisms contribute to the correct delivery of these cargos to their target domains. One mechanism is through motor recognition of sub-domain-specific microtubule (MT) tracks, sign-posted by different tubulin isoforms, tubulin post-translational modifications, tubulin GTPase activity and MT-associated proteins (MAPs). With neurons as a model system, a critical review of these regulatory mechanisms is presented here, with a particular focus on the emerging contribution of compartmentalised MAPs. Overall, we conclude that – especially for axonal cargo – alterations to the MT track can influence transport, although in vivo, it is likely that multiple track-based effects act synergistically to ensure accurate cargo distribution.

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Nadia Bahi-Buisson, Isabelle Souville, Franck J Fourniol, Aurelie Toussaint, Carolyn A Moores, Anne Houdusse, Jean Yves Lemaitre, Karine Poirier, Reham Khalaf-Nazzal, Marie Hully, Pierre Louis Leger, Caroline Elie, Nathalie Boddaert, Cherif Beldjord, Jamel Chelly, Fiona Francis, (2013 Feb 1)

New insights into genotype-phenotype correlations for the doublecortin-related lissencephaly spectrum.

Brain : a journal of neurology : 223-44 : DOI : 10.1093/brain/aws323 En savoir plus
Résumé

X-linked isolated lissencephaly sequence and subcortical band heterotopia are allelic human disorders associated with mutations of doublecortin (DCX), giving both familial and sporadic forms. DCX encodes a microtubule-associated protein involved in neuronal migration during brain development. Structural data show that mutations can fall either in surface residues, likely to impair partner interactions, or in buried residues, likely to impair protein stability. Despite the progress in understanding the molecular basis of these disorders, the prognosis value of the location and impact of individual DCX mutations has largely remained unclear. To clarify this point, we investigated a cohort of 180 patients who were referred with the agyria-pachygyria subcortical band heterotopia spectrum. DCX mutations were identified in 136 individuals. Analysis of the parents’ DNA revealed the de novo occurrence of DCX mutations in 76 cases [62 of 70 females screened (88.5%) and 14 of 60 males screened (23%)], whereas in the remaining cases, mutations were inherited from asymptomatic (n = 14) or symptomatic mothers (n = 11). This represents 100% of families screened. Female patients with DCX mutation demonstrated three degrees of clinical-radiological severity: a severe form with a thick band (n = 54), a milder form (n = 24) with either an anterior thin or an intermediate thickness band and asymptomatic carrier females (n = 14) with normal magnetic resonance imaging results. A higher proportion of nonsense and frameshift mutations were identified in patients with de novo mutations. An analysis of predicted effects of missense mutations showed that those destabilizing the structure of the protein were often associated with more severe phenotypes. We identified several severe- and mild-effect mutations affecting surface residues and observed that the substituted amino acid is also critical in determining severity. Recurrent mutations representing 34.5% of all DCX mutations often lead to similar phenotypes, for example, either severe in sporadic subcortical band heterotopia owing to Arg186 mutations or milder in familial cases owing to Arg196 mutations. Taken as a whole, these observations demonstrate that DCX-related disorders are clinically heterogeneous, with severe sporadic and milder familial subcortical band heterotopia, each associated with specific DCX mutations. There is a clear influence of the individual mutated residue and the substituted amino acid in determining phenotype severity.

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Année de publication : 2012

Judy S Liu, Christian R Schubert, Xiaoqin Fu, Franck J Fourniol, Jyoti K Jaiswal, Anne Houdusse, Collin M Stultz, Carolyn A Moores, Christopher A Walsh (2012 Aug 4)

Molecular basis for specific regulation of neuronal kinesin-3 motors by doublecortin family proteins.

Molecular cell : 707-21 : DOI : 10.1016/j.molcel.2012.06.025 En savoir plus
Résumé

Doublecortin (Dcx) defines a growing family of microtubule (MT)-associated proteins (MAPs) involved in neuronal migration and process outgrowth. We show that Dcx is essential for the function of Kif1a, a kinesin-3 motor protein that traffics synaptic vesicles. Neurons lacking Dcx and/or its structurally conserved paralogue, doublecortin-like kinase 1 (Dclk1), show impaired Kif1a-mediated transport of Vamp2, a cargo of Kif1a, with decreased run length. Human disease-associated mutations in Dcx’s linker sequence (e.g., W146C, K174E) alter Kif1a/Vamp2 transport by disrupting Dcx/Kif1a interactions without affecting Dcx MT binding. Dcx specifically enhances binding of the ADP-bound Kif1a motor domain to MTs. Cryo-electron microscopy and subnanometer-resolution image reconstruction reveal the kinesin-dependent conformational variability of MT-bound Dcx and suggest a model for MAP-motor crosstalk on MTs. Alteration of kinesin run length by MAPs represents a previously undiscovered mode of control of kinesin transport and provides a mechanism for regulation of MT-based transport by local signals.

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