Publications de l’équipe
Année de publication : 2019
Plasmodium myosin A drives parasite invasion by an atypical force generating mechanism.
Nature communications : 3286 : DOI : 10.1038/s41467-019-11120-0 En savoir plusRésumé
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.
ReplierAnnée de publication : 2018
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 plusRésumé
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.
ReplierCircularly Permuted Fluorogenic Proteins for the Design of Modular Biosensors.
ACS chemical biology : DOI : 10.1021/acschembio.8b00417 En savoir plusRésumé
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.
ReplierMyosin 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 plusRésumé
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.
ReplierAn 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 plusRésumé
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.
ReplierAnnée de publication : 2017
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 plusRésumé
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.
ReplierEml1 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 plusRésumé
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.
ReplierCoupling 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 plusRésumé
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.
ReplierThe divergent mitotic kinesin MKLP2 exhibits atypical structure and mechanochemistry.
eLife : DOI : 10.7554/eLife.27793 En savoir plusRésumé
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.
ReplierMechanistic 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 plusRésumé
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.
ReplierMyosin 7 and its adaptors link cadherins to actin.
Nature communications : 15864 : DOI : 10.1038/ncomms15864 En savoir plusRésumé
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.
ReplierRab GTPases and their interacting protein partners: structural insights into Rab functional diversity.
Small GTPases : 0 : DOI : 10.1080/21541248.2017.1336191 En savoir plusRésumé
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.
ReplierEmerging 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 plusRésumé
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.
ReplierOxidation of F-actin controls the terminal steps of cytokinesis.
Nature communications : 14528 : DOI : 10.1038/ncomms14528 En savoir plusRésumé
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.
ReplierAnnée de publication : 2016
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 plusRésumé
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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