ALS-linked PFN1 variants exhibit loss and gain of functions in the context of formin-induced actin polymerization [LINK]Eric J. Schmidt, Salome Funes, Jeanne E. McKeon, Brittany R. Morgan, Sivakumar Boopathy, Lauren C. O’Connor, Osman Bilsel, Francesca Massi, Antoine Jégou & Daryl A. BoscoPNAS 2021
ALS-linked PFN1 variants exhibit loss and gain of functions in the context of formin-induced actin polymerization [LINK]
Abstract : Profilin-1 (PFN1) plays important roles in modulating actin dynamics through binding both monomeric actin and proteins enriched with polyproline motifs. Mutations in PFN1 have been linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). However, whether ALS-linked mutations affect PFN1 function has remained unclear. To address this question, we employed an unbiased proteomics analysis in mammalian cells to identify proteins that differentially interact with mutant and wild-type (WT) PFN1. These studies uncovered differential binding between two ALS-linked PFN1 variants, G118V and M114T, and select formin proteins. Furthermore, both variants augmented formin-mediated actin assembly relative to PFN1 WT. Molecular dynamics simulations revealed mutation-induced changes in the internal dynamic couplings within an alpha helix of PFN1 that directly contacts both actin and polyproline, as well as structural fluctuations within the actin- and polyproline-binding regions of PFN1. These data indicate that ALS-PFN1 variants have the potential for heightened flexibility in the context of the ternary actin-PFN1-polyproline complex during actin assembly. Conversely, PFN1 C71G was more severely destabilized than the other PFN1 variants, resulting in reduced protein expression in both transfected and ALS patient lymphoblast cell lines. Moreover, this variant exhibited loss-of-function phenotypes in the context of actin assembly. Perturbations in actin dynamics and assembly can therefore result from ALS-linked mutations in PFN1. However, ALS-PFN1 variants may dysregulate actin polymerization through different mechanisms that depend upon the solubility and stability of the mutant protein.
The dynamic instability of actin filament barbed ends [LINK]Guillaume Romet-Lemonne & Antoine JegouJournal of Cell Biology 2021 - Viewpoint
The dynamic instability of actin filament barbed ends [LINK]
Abstract : The turnover of actin filament networks in cells has long been considered to reflect the treadmilling behavior of pure actin filaments in vitro, where only the pointed ends depolymerize. Newly discovered molecular mechanisms challenge this notion, as they provide evidence of situations in which growing and depolymerizing barbed ends coexist.
Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks [LINK]Markku Hakala, Hugo Wioland, Mari Tolonen, Tommi Kotila, Antoine Jegou, Guillaume Romet-Lemonne & Pekka LappalainenNature Cell Biology 2021
Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks [LINK]
Abstract : Coordinated polymerization of actin filaments provides force for cell migration, morphogenesis and endocytosis. Capping protein (CP) is a central regulator of actin dynamics in all eukaryotes. It binds to actin filament (F-actin) barbed ends with high affinity and slow dissociation kinetics to prevent filament polymerization and depolymerization. However, in cells, CP displays remarkably rapid dynamics within F-actin networks, but the underlying mechanism remains unclear. Here, we report that the conserved cytoskeletal regulator twinfilin is responsible for CP’s rapid dynamics and specific localization in cells. Depletion of twinfilin led to stable association between CP and cellular F-actin arrays, as well as to its retrograde movement throughout leading-edge lamellipodia. These were accompanied by diminished F-actin turnover rates. In vitro single-filament imaging approaches revealed that twinfilin directly promotes dissociation of CP from filament barbed ends, while enabling subsequent filament depolymerization. These results uncover a bipartite mechanism that controls how actin cytoskeleton-mediated forces are generated in cells.
Actin filament oxidation by MICAL1 suppresses protections from cofilin-induced disassembly [LINK]Hugo Wioland, Stéphane Frémont, Bérengère Guichard, Arnaud Echard, Antoine Jégou* & Guillaume Romet-Lemonne*EMBO Reports 2021
Actin filament oxidation by MICAL1 suppresses protections from cofilin-induced disassembly [LINK]
Abstract : Proteins of the ADF/cofilin family play a central role in the disassembly of actin filaments, and their activity must be tightly regulated in cells. Recently, the oxidation of actin filaments by the enzyme MICAL1 was found to amplify the severing action of cofilin through unclear mechanisms. Using single filament experiments in vitro, we found that actin filament oxidation by MICAL1 increases, by several orders of magnitude, both cofilin binding and severing rates, explaining the dramatic synergy between oxidation and cofilin for filament disassembly. Remarkably, we found that actin oxidation bypasses the need for cofilin activation by dephosphorylation. Indeed, non‐activated, phosphomimetic S3D‐cofilin binds and severs oxidized actin filaments rapidly, in conditions where non‐oxidized filaments are unaffected. Finally, tropomyosin Tpm1.8 loses its ability to protect filaments from cofilin severing activity when actin is oxidized by MICAL1. Together, our results show that MICAL1‐induced oxidation of actin filaments suppresses their physiological protection from the action of cofilin. We propose that, in cells, direct post‐translational modification of actin filaments by oxidation is a way to trigger their disassembly.
Antoine Jégou* & Guillaume Romet-Lemonne*Current Opinion in Cell Biology 2021 - Review
Abstract : In cells, the actin cytoskeleton is regulated by an interplay between mechanics and biochemistry. A key mechanism, which has emerged based on converging indications from structural, cellular, and biophysical data, depicts the actin filament as a mechanically tunable substrate: mechanical stress applied to an actin filament induces conformational changes, which modify the binding and the regulatory action of actin-binding proteins. For a long time, however, direct evidence of this mechanotransductive mechanism was very scarce. This situation is changing rapidly, and recent in vitro single-filament studies using different techniques have revealed that several actin-binding proteins are able to sense tension, curvature, and/or torsion, applied to actin filaments. Here, we discuss these recent advances and their possible implications.
The Arp1/11 Minifilament of Dynactin Primes the Endosomal Arp2/3 Complex [LINK]Artem I. Fokin, Violaine David, Ksenia Oguievetskaia, Emmanuel Derivery, Caroline E. Stone, Luyan Cao, Nathalie Rocques, Nicolas Molinie, Véronique Henriot, Magali Aumont-Nicaise, Maria-Victoria Hinckelmann, Frédéric Saudou, Christophe Le Clainche, Andrew P. Carter, Guillaume Romet-Lemonne & Alexis M. Gautreau. Science Advances 2021
The Arp1/11 Minifilament of Dynactin Primes the Endosomal Arp2/3 Complex [LINK]
Abstract : Dendritic actin networks develop from a first actin filament through branching by the Arp2/3 complex. At the surface of endosomes, the WASH complex activates the Arp2/3 complex and interacts with the Capping Protein for unclear reasons. Here we show that that the WASH complex interacts with Dynactin and uncaps it through its FAM21 subunit. In vitro, the uncapped Arp1/11 minifilament elongates an actin filament, which then primes the WASH-induced Arp2/3 branching reaction. In Dynactin-depleted cells or in cells where the WASH complex is reconstituted with a FAM21 mutant that cannot uncap Dynactin, formation of branched actin at the endosomal surface is impaired. Our results reveal the importance of the WASH complex in coordinating two complexes containing actin-related proteins.
SPIN90 associates with mDia1 and the Arp2/3 complex to regulate cortical actin organizationLuyan Cao, Amina Yonis, Malti Vaghela, Elias H. Barriga, Priyamvada Chugh, Matthew B. Smith, Julien Maufront, Geneviève Lavoie, Antoine Méant, Emma Ferber, Miia Bovellan, Art Alberts, Aurélie Bertin, Roberto Mayor, Ewa K. Paluch, Philippe P. Roux, Antoine Jégou*, Guillaume Romet-Lemonne* & Guillaume Charras*Nature Cell Biology 2020
SPIN90 associates with mDia1 and the Arp2/3 complex to regulate cortical actin organization
Abstract : Cell shape is controlled by the submembranous cortex, an actomyosin network mainly generated by two actin nucleators: the Arp2/3 complex and the formin mDia1. Changes in relative nucleator activity may alter cortical organization, mechanics and cell shape. Here we investigate how nucleation-promoting factors mediate interactions between nucleators. In vitro, the nucleation-promoting factor SPIN90 promotes formation of unbranched filaments by Arp2/3, a process thought to provide the initial filament for generation of dendritic networks. Paradoxically, in cells, SPIN90 appears to favour a formin-dominated cortex. Our in vitro experiments reveal that this feature stems mainly from two mechanisms: efficient recruitment of mDia1 to SPIN90-Arp2/3 nucleated filaments and formation of a ternary SPIN90-Arp2/3-mDia1 complex that greatly enhances filament nucleation. Both mechanisms yield rapidly elongating filaments with mDia1 at their barbed ends and SPIN90-Arp2/3 at their pointed ends. Thus, in networks, SPIN90 lowers branching densities and increases the proportion of long filaments elongated by mDia1.
Dynamics of Tpm1.8 domains on actin filaments with single molecule resolution [PDF LINK]Ilina Bareja, Hugo Wioland, Miro Janco, Philip R. Nicovich, Antoine Jégou, Guillaume Romet-Lemonne, James Walsh & Till Böcking.Mol Biol Cell 2020
Dynamics of Tpm1.8 domains on actin filaments with single molecule resolution [PDF LINK]
Abstract : Tropomyosins regulate dynamics and functions of the actin cytoskeleton by forming long chains along the two strands of actin filaments that act as gatekeepers for the binding of other actin-binding proteins. The fundamental molecular interactions underlying the binding of tropomyosin to actin are still poorly understood. Using microfluidics and fluorescence microscopy, we observed the binding of fluorescently labelled tropomyosin isoform Tpm1.8 to unlabelled actin filaments in real time. This approach in conjunction with mathematical modeling enabled us to quantify the nucleation, assembly and disassembly kinetics of Tpm1.8 on single filaments and at the single molecule level. Our analysis suggests that Tpm1.8 decorates the two strands of the actin filament independently. Nucleation of a growing tropomyosin domain proceeds with high probability as soon as the first Tpm1.8 molecule is stabilised by the addition of a second molecule, ultimately leading to full decoration of the actin filament. In addition, Tpm1.8 domains are asymmetrical, with enhanced dynamics at the edge oriented towards the barbed end of the actin filament. The complete description of Tpm1.8 kinetics on actin filaments presented here provides molecular insight into actin-tropomyosin filament formation and the role of tropomyosins in regulating actin filament dynamics.
Ottilie Von Loeffelholz, Andrew Purkiss, Luyan Cao, Svend Kjaer, Naoko Kogata, Guillaume Romet-Lemonne, Michael Way, Carolyn A Moores .Biology Open 2020
Abstract : The Arp2/3 complex regulates many cellular processes by stimulating formation of branched actin filament networks. Because three of its seven subunits exist as two different isoforms, mammals produce a family of Arp2/3 complexes with different properties that may be suited to different physiological contexts. To shed light on how isoform diversification affects Arp2/3 function, we determined a 4.2 Å resolution cryo-EM structure of the most active human Arp2/3 complex containing ARPC1B and ARPC5L, and compared it with the structure of the least active ARPC1A-ARPC5-containing complex. The architecture of each isoform-specific Arp2/3 complex is the same. Strikingly, however, the N-terminal half of ARPC5L is partially disordered compared to ARPC5, suggesting that this region of ARPC5/ARPC5L is an important determinant of complex activity. Confirming this idea, the nucleation activity of Arp2/3 complexes containing hybrid ARPC5/ARPC5L subunits is higher when the ARPC5L N-terminus is present, thereby providing insight into activity differences between the different Arp2/3 complexes.
In vitro reconstitution of dynein force exertion in bulk cytoplasm [PDF LINK]Héliciane Palenzuela, Benjamin Lacroix , Jérémy Sallé , Katsuhiko Minami , Tomohiro Shima , Antoine Jégou, Guillaume Romet-Lemonne* & Nicolas Minc* Current Biology 2020
In vitro reconstitution of dynein force exertion in bulk cytoplasm [PDF LINK]
Abstract : The forces generated by Microtubules (MTs) and their associated motors orchestrate essential cellular processes ranging from vesicular trafficking to centrosome positioning. To date, most studies have focused on force exertion from motors anchored on a static surface, such as the cell cortex in vivo or glass surfaces in vitro. However, motors also transport large cargos and endomembrane networks, whose hydrodynamic interactions with the viscous cytoplasm should generate sizable forces in bulk. Such forces may contribute to MT aster centration, organization and orientation, but have yet to be evidenced and studied in a minimal reconstituted system. By developing a bulk motility assay, based on stabilized MTs and dynein-coated beads freely floating in a viscous medium away from any surface, we demonstrate that the motion of a cargo exerts a pulling force on the MT and propels it in opposite direction. Quantification of resulting MT movements for different motors, motor velocities, over a range of cargo size and medium viscosities, shows that the efficiency of this mechanism is primarily determined by cargo size and MT length. Forces exerted by cargos are additive, allowing us to recapitulate tug-of-war situations, or bi-dimensional motions of minimal asters. These data also reveal unappreciated effects of the nature of viscous crowders and hydrodynamic interactions between cargos and MTs, likely relevant to understand this mode of force exertion in living cells. This study places endomembrane transport as a significant mode of MT force exertion with far-reaching consequences for cellular organization.
Abstract : Abscission is the terminal step of cytokinesis leading to the physical separation of the daughter cells. In response to the abnormal presence of lagging chromatin between dividing cells, an evolutionarily conserved abscission/NoCut checkpoint delays abscission and prevents formation of binucleated cells by stabilizing the cytokinetic intercellular bridge (ICB). How this bridge is stably maintained for hours while the checkpoint is activated is poorly understood and has been proposed to rely on F-actin in the bridge region. Here, we show that actin polymerization is indeed essential for stabilizing the ICB when lagging chromatin is present, but not in normal dividing cells. Mechanistically, we found that a cytosolic pool of human methionine sulfoxide reductase B2 (MsrB2) is strongly recruited at the midbody in response to the presence of lagging chromatin and functions within the ICB to promote actin polymerization there. Consistently, in MsrB2-depleted cells, F-actin levels are decreased in ICBs, and dividing cells with lagging chromatin become binucleated as a consequence of unstable bridges. We further demonstrate that MsrB2 selectively reduces oxidized actin monomers and thereby counteracts MICAL1, an enzyme known to depolymerize actin filaments by direct oxidation. Finally, MsrB2 colocalizes and genetically interacts with the checkpoint components Aurora B and ANCHR, and the abscission delay upon checkpoint activation by nuclear pore defects also depends on MsrB2. Altogether, this work reveals that actin reduction by MsrB2 is a key component of the abscission checkpoint that favors F-actin polymerization and limits tetraploidy, a starting point for tumorigenesis.
Geometrical constraints greatly hinder formin mDia1 activity [PDF LINK]Emiko L. Suzuki, Jahnavi Chikireddy, Serge Dmitrieff, Bérengère Guichard, Guillaume Romet-Lemonne* & Antoine Jégou*Nano Letters 2020
Geometrical constraints greatly hinder formin mDia1 activity [PDF LINK]
Abstract : Formins are one of the central players in the assembly of most actin networks in cells. The sensitivity of these processive molecular machines to mechanical tension is now well established. However, how the activity of formins is affected by geometrical constraints related to network architecture, such as filament cross-linking and formin spatial confinement, remains largely unknown. Combining microfluidics and micropatterning, we reconstituted in vitro mDia1 formin-elongated filament bundles induced by fascin, with different geometrical constraints on the formins, and measured the impact of these constraints on formin elongation rate and processivity. When filaments are not bundled, the anchoring details of formins have only a mild impact on their processivity and do not affect their elongation rate. When formins are unanchored, we show that filament bundling by fascin reduces both their elongation rate and their processivity. Strikingly, when filaments elongated by surface-anchored formins are cross-linked together, formin elongation rate immediately decreases and processivity is reduced up to 24-fold depending on the cumulative impact of formin rotational and translational freedom. Our results reveal an unexpected crosstalk between the constraints at the filament and the formin levels. We anticipate that in cells the molecular details of formin anchoring to the plasma membrane strongly modulate formin activity at actin filament barbed ends.
The many implications of actin filament helicity [PDF LINK]Antoine Jégou & Guillaume Romet-LemonneSeminars in Cell and Developmental Biology 2020 - Review
The many implications of actin filament helicity [PDF LINK]
Abstract : One of the best known features of actin filaments is their helical structure. A number of essential properties emerge from this molecular arrangement of actin subunits. Here, we give an overview of the mechanical and biochemical implications of filament helicity, at different scales. In particular, a number of recent studies have highlighted the role of filament helicity in the adaptation to and the generation of mechanical torsion, and in the modulation of the filament’s interaction with very different actin-binding proteins (such as myosins, cross-linkers, formins, and cofilin). Helicity can thus be seen as a key factor for the regulation of actin assembly, and as a link between biochemical regulators and their mechanical context. In addition, actin filament helicity appears to play an essential role in the establishment of chirality at larger scales, up to the organismal scale. Altogether, helicity appears to be an essential feature contributing to the regulation of actin assembly dynamics, and to actin’s ability to organize cells at a larger scale.
The advantages of microfluidics to study actin biochemistry and biomechanics [PDF LINK]Hugo Wioland, Emiko L Suzuki, Luyan Cao, Guillaume Romet-Lemonne* & Antoine Jégou*J Muscle Res Cell Motil 2020 - Methods review
The advantages of microfluidics to study actin biochemistry and biomechanics [PDF LINK]
Abstract : The regulated assembly of actin filaments is essential in nearly all cell types. Studying actin assembly dynamics can pose many technical challenges. A number of these challenges can be overcome by using microfluidics to observe and manipulate single actin filaments under an optical microscope. In particular, microfluidics can be tremendously useful for applying different mechanical stresses to actin filaments and determining how the physical context of the filaments affects their regulation by biochemical factors. In this review, we summarize the main features of microfluidics for the study of actin assembly dynamics, and we highlight some recent developments that have emerged from the combination of microfluidics and other techniques. We use two case studies to illustrate our points: the rapid assembly of actin filaments by formins and the disassembly of filaments by actin depolymerizing factor (ADF)/cofilin. Both of these protein families play important roles in cells. They regulate actin assembly through complex molecular mechanisms that are sensitive to the filaments’ mechanical context, with multiple activities that need to be quantified separately. Microfluidics-based experiments have been extremely useful for gaining insight into the regulatory actions of these two protein families.
Mechanism of synergistic actin filament pointed end depolymerization by cyclase-associated protein and cofilin [PDF LINK]Tommi Kotila, Hugo Wioland, Giray Enkavi, Konstantin Kogan, Ilpo Vattulainen, Antoine Jégou, Guillaume Romet-Lemonne & Pekka LappalainenNature Communications 2019 10:5320
Mechanism of synergistic actin filament pointed end depolymerization by cyclase-associated protein and cofilin [PDF LINK]
Abstract : The ability of cells to generate forces through actin filament turnover was an early adaptation in evolution. While much is known about how actin filaments grow, mechanisms of their disassembly are incompletely understood. The best-characterized actin disassembly factors are the cofilin family proteins, which increase cytoskeletal dynamics by severing actin filaments. However, the mechanism by which severed actin filaments are recycled back to monomeric form has remained enigmatic. We report that cyclase-associated-protein (CAP) works in synergy with cofilin to accelerate actin filament depolymerization by nearly 100-fold. Structural work uncovers the molecular mechanism by which CAP interacts with actin filament pointed end to destabilize the interface between terminal actin subunits, and subsequently recycles the newly-depolymerized actin monomer for the next round of filament assembly. These findings establish CAP as a molecular machine promoting rapid actin filament depolymerization and monomer recycling, and explain why CAP is critical for actin-dependent processes in all eukaryotes.
Myosin IIA and formin dependent mechanosensitivity of filopodia adhesion [PDF LINK] N. O. Alieva, A. K. Efremov, S. Hu, D. Oh, Z. Chen, M. Natarajan, H. T. Ong, , A. Jegou, G. Romet-Lemonne, J. T. Groves, M. P. Sheetz, J. Yan & A. D. BershadskyNature Communications 2019 10:3593
Myosin IIA and formin dependent mechanosensitivity of filopodia adhesion [PDF LINK]
Abstract : Filopodia, dynamic membrane protrusions driven by polymerization of an actin filament core, can adhere to the extracellular matrix and experience both external and cell-generated pulling forces. The role of such forces in filopodia adhesion is however insufficiently understood. Here, we study filopodia induced by overexpression of myosin X, typical for cancer cells. The lifetime of such filopodia positively correlates with the presence of myosin IIA filaments at the filopodia bases. Application of pulling forces to the filopodia tips through attached fibronectin-coated laser-trapped beads results in sustained growth of the filopodia. Pharmacological inhibition or knockdown of myosin IIA abolishes the filopodia adhesion to the beads. Formin inhibitor SMIFH2, which causes detachment of actin filaments from formin molecules, produces similar effect. Thus, centripetal force generated by myosin IIA filaments at the base of filopodium and transmitted to the tip through actin core in a formin-dependent fashion is required for filopodia adhesion.
Torsional stress generated by ADF/cofilin on cross-linked actin filaments boosts their severing [PDF LINK]Hugo Wioland, Antoine Jegou*, Guillaume Romet-Lemonne*P.N.A.S. 2019 online
Torsional stress generated by ADF/cofilin on cross-linked actin filaments boosts their severing [PDF LINK]
Abstract : Proteins of the actin depolymerizing factor (ADF)/cofilin family are the central regulators of actin filament disassembly. A key function of ADF/cofilin is to sever actin filaments. However, how it does so in a physiological context, where filaments are interconnected and under mechanical stress, remains unclear. Here, we monitor and quantify the action of ADF/cofilin in different mechanical situations by using single-molecule, single-filament, and filament network techniques, coupled to microfluidics. We find that local curvature favors severing, while tension surprisingly has no effect on cofilin binding and weakly enhances severing. Remarkably, we observe that filament segments that are held between two anchoring points, thereby constraining their twist, experience a mechanical torque upon cofilin binding. We find that this ADF/cofilin-induced torque does not hinder ADF/cofilin binding, but dramatically enhances severing. A simple model, which faithfully recapitulates our experimental observations, indicates that the ADF/cofilin-induced torque increases the severing rate constant 100-fold. A consequence of this mechanism, which we verify experimentally, is that cross-linked filament networks are severed by cofilin far more efficiently than nonconnected filaments. We propose that this mechanochemical mechanism is critical to boost ADF/cofilin’s ability to sever highly connected filament networks in cells.
Quantitative Variations with pH of Actin Depolymerizing Factor/Cofilin's Multiple Actions on Actin Filaments [PDF LINK]Hugo Wioland, Antoine Jégou*, Guillaume Romet-Lemonne*Biochemistry. 2019 58(1):40-47
Quantitative Variations with pH of Actin Depolymerizing Factor/Cofilin's Multiple Actions on Actin Filaments [PDF LINK]
Abstract : Actin depolymerizing factor (ADF)/cofilin is the main protein family promoting the disassembly of actin filaments, which is essential for numerous cellular functions. ADF/cofilin proteins disassemble actin filaments through different reactions, as they bind to their sides, sever them, and promote the depolymerization of the resulting ADF/cofilin-saturated filaments. Moreover, the efficiency of ADF/cofilin is known to be very sensitive to pH. ADF/cofilin thus illustrates two challenges in actin biochemistry: separating the different regulatory actions of a single protein and characterizing them as a function of specific biochemical conditions. Here, we investigate the different reactions of ADF/cofilin on actin filaments, at four different pH values ranging from 6.6 to 7.8, using single-filament microfluidics techniques. We show that decreasing the pH decreases the effective filament severing rate by increasing the rate at which filaments become saturated by ADF/cofilin, thereby reducing the number of ADF/cofilin domain boundaries, where severing can occur. The severing rate per domain boundary, however, remains unchanged at different pH values. The ADF/cofilin-decorated filaments ("cofilactin" filaments) depolymerize from both ends. We show here that, at physiological pH (7.0-7.4), the pointed end depolymerization of cofilactin filaments is barely faster than that of bare filaments. In contrast, cofilactin barbed ends undergo an "unstoppable" depolymerization (depolymerizing for minutes despite the presence of free actin monomers and capping protein in solution), throughout our pH range. We thus show that, at physiological pH, the main contribution of ADF/cofilin to filament depolymerization is at the barbed end.
Using microfluidics single filament assay to study formin control of actin assemblyGuillaume Romet-Lemonne, Bérengère Guichard, Antoine JégouMethods Mol Biol 2018 1085:75-92 - Methods
Using microfluidics single filament assay to study formin control of actin assembly
Abstract : Formin is a highly processive motor that offers very unique features to control the elongation of actin filaments. When bound to the filament barbed-end, it enhances the addition of profilin-actin from solution to dramatically accelerate actin assembly. The different aspects of formin activity can be explored using single actin filament assays based on the combination of microfluidics with fluorescence microscopy. This chapter describes methods to conduct single filament experiments and explains how to probe formin renucleation as a case study: purification of the proteins, the design, preparation, and assembly of the flow chamber, and how to specifically anchor formins to the surface.
Modulation of formin processivity by profilin and mechanical tension [PDF LINK]Luyan Cao, Mikael Kerleau, Emiko L. Suzuki, Hugo Wioland, Sandy Jouet, Bérengère Guichard, Martin Lenz, Guillaume Romet-Lemonne*, Antoine Jégou*
Modulation of formin processivity by profilin and mechanical tension [PDF LINK]
eLife 2018 DOI: 10.7554/eLife.34176
Abstract : Formins are major regulators of actin networks. They enhance actin filament dynamics by remaining processively bound to filament barbed ends. How biochemical and mechanical factors affect formin processivity are open questions. Monitoring individual actin filaments in a microfluidic flow, we report that formins mDia1 and mDia2 dissociate faster under higher ionic strength and when actin concentration is increased. Profilin, known to increase the elongation rate of formin-associated filaments, surprisingly decreases the formin dissociation rate, by bringing formin FH1 domains in transient contact with the barbed end. In contrast, piconewton tensile forces applied to actin filaments accelerate formin dissociation by orders of magnitude, largely overcoming profilin-mediated stabilization. We developed a model of formin conformations showing that our data indicates the existence of two different dissociation pathways, with force favoring one over the other. How cells limit formin dissociation under tension is now a key question for future studies.
ADF/Cofilin Accelerates Actin Dynamics by Severing Filaments and Promoting Their Depolymerization at Both Ends [PDF LINK]Hugo Wioland, Berengere Guichard, Yosuke Senju, Sarah Myram, Pekka Lappalainen, Antoine Jegou* & Guillaume Romet-Lemonne*Current Biology 2017 130: 1509-1517
ADF/Cofilin Accelerates Actin Dynamics by Severing Filaments and Promoting Their Depolymerization at Both Ends [PDF LINK]
Abstract : Actin-depolymerizing factor (ADF)/cofilins contribute to cytoskeletal dynamics by promoting rapid actin filament disassembly. In the classical view, ADF/cofilin sever filaments, and capping proteins block filament barbed ends whereas pointed ends depolymerize, at a rate that is still debated. Here, by monitoring the activity of the three mammalian ADF/cofilin isoforms on individual skeletal muscle and cytoplasmic actin filaments, we directly quantify the reactions underpinning filament severing and depolymerization from both ends. We find that, in the absence of monomeric actin, soluble ADF/cofilin can associate with bare filament barbed ends to accelerate their depolymerization. Compared to bare filaments, ADF/cofilin-saturated filaments depolymerize faster from their pointed ends and slower from their barbed ends, resulting in similar depolymerization rates at both ends. This effect is isoform specific because depolymerization is faster for ADF- than for cofilin-saturated filaments. We also show that, unexpectedly, ADF/cofilin-saturated filaments qualitatively differ from bare filaments: their barbed ends are very difficult to cap or elongate, and consequently undergo depolymerization even in the presence of capping protein and actin monomers. Such depolymerizing ADF/cofilin-decorated barbed ends are produced during 17% of severing events. They are also the dominant fate of filament barbed ends in the presence of capping protein, because capping allows growing ADF/cofilin domains to reach the barbed ends, thereby promoting their uncapping and subsequent depolymerization. Our experiments thus reveal how ADF/cofilin, together with capping protein, control the dynamics of actin filament barbed and pointed ends. Strikingly, our results propose that significant barbed-end depolymerization may take place in cells.
Emerging roles of MICAL family proteins – from actin oxidation to membrane trafficking during cytokinesis [PDF LINK]Stéphane Frémont, Guillaume Romet-Lemonne, Anne Houdusse & Arnaud EchardJournal of Cell Science 2017 130: 1509-1517 - Review
Emerging roles of MICAL family proteins – from actin oxidation to membrane trafficking during cytokinesis [PDF LINK]
Abstract : 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.
Oxidation of F-actin controls the terminal steps of cytokinesis [PDF LINK]Stéphane Frémont, Hussein Hammich, Jian Bai, Hugo Wioland, Kerstin Klinkert, Murielle Rocancourt, Carlos Kikuti, David Stroebel, Guillaume Romet-Lemonne, Olena Pylypenko, Anne Houdusse & Arnaud EchardNature Communications 2017 8:14528
Oxidation of F-actin controls the terminal steps of cytokinesis [PDF LINK]
Abstract : 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.
Single Filaments to Reveal the Multiple Flavors of Actin [PDF LINK]Antoine Jégou & Guillaume Romet-LemonneBiophysical Journal 110(10) 2238 - Review
Single Filaments to Reveal the Multiple Flavors of Actin [PDF LINK]
Abstract : A number of key cell processes rely on specific assemblies of actin filaments, which are all constructed from nearly identical building blocks: the abundant and extremely conserved actin protein. A central question in the field is to understand how different filament networks can coexist and be regulated. Discoveries in science are often related to technical advances. Here, we focus on the ongoing single filament revolution and discuss how these techniques have greatly contributed to our understanding of actin assembly. In particular, we highlight how they have refined our understanding of the many protein-based regulatory mechanisms that modulate actin assembly. It is now becoming apparent that other factors give filaments a specific identity that determines which proteins will bind to them. We argue that single filament techniques will play an essential role in the coming years as we try to understand the many ways actin filaments can take different flavors and unveil how these flavors modulate the action of regulatory proteins. We discuss different factors known to make actin filaments distinguishable by regulatory proteins and speculate on their possible consequences.
Formin and capping protein together embrace the actin filament in a ménage à trois [PDF LINK] Shashank Shekhar, Mikael Kerleau, Sonja Kühn, Julien Pernier, Guillaume Romet-Lemonne, Antoine Jégou* & Marie-France Carlier*Nature Communications 2015 4:8730
Formin and capping protein together embrace the actin filament in a ménage à trois [PDF LINK]
Abstract : Proteins targeting actin filament barbed ends play a pivotal role in motile processes. While formins enhance filament assembly, capping protein (CP) blocks polymerization. On their own, they both bind barbed ends with high affinity and very slow dissociation. Their barbed-end binding is thought to be mutually exclusive. CP has recently been shown to be present in filopodia and controls their morphology and dynamics. Here we explore how CP and formins may functionally coregulate filament barbed-end assembly. We show, using kinetic analysis of individual filaments by microfluidics-assisted fluorescence microscopy, that CP and mDia1 formin are able to simultaneously bind barbed ends. This is further confirmed using single-molecule imaging. Their mutually weakened binding enables rapid displacement of one by the other. We show that formin FMNL2 behaves similarly, thus suggesting that this is a general property of formins. Implications in filopodia regulation and barbed-end structural regulation are discussed.
Cellular control of cortical actin nucleationBovelaan M, Romero Y, Biro M, Fritzsche M, Boden A, Moulding D, Thorogate R, Jégou A., Thrasher A, Romet-Lemonne G, Paluch E, Roux PP and Charras GCurrent Biology 2014 ; 24(14): 1628-35.
Cellular control of cortical actin nucleation
Abstract : The contractile actin cortex is a thin layer of actin, myosin, and actin-binding proteins that subtends the membrane of animal cells. The cortex is the main determinant of cell shape and plays a fundamental role in cell division [1-3], migration , and tissue morphogenesis . For example, cortex contractility plays a crucial role in amoeboid migration of metastatic cells  and during division, where its misregulation can lead to aneuploidy . Despite its importance, our knowledge of the cortex is poor, and even the proteins nucleating it remain unknown, though a number of candidates have been proposed based on indirect evidence [8-15]. Here, we used two independent approaches to identify cortical actin nucleators: a proteomic analysis using cortex-rich isolated blebs, and a localization/small hairpin RNA (shRNA) screen searching for phenotypes with a weakened cortex or altered contractility. This unbiased study revealed that two proteins generated the majority of cortical actin: the formin mDia1 and the Arp2/3 complex. Each nucleator contributed a similar amount of F-actin to the cortex but had very different accumulation kinetics. Electron microscopy examination revealed that each nucleator affected cortical network architecture differently. mDia1 depletion led to failure in division, but Arp2/3 depletion did not. Interestingly, despite not affecting division on its own, Arp2/3 inhibition potentiated the effect of mDia1 depletion. Our findings indicate that the bulk of the actin cortex is nucleated by mDia1 and Arp2/3 and suggest a mechanism for rapid fine-tuning of cortex structure and mechanics by adjusting the relative contribution of each nucleator.
Actin filament dynamics using microfluidics Carlier MF, Romet-Lemonne G, Jégou AMethods Enzymol. 2014 ; 540:3-17.
Actin filament dynamics using microfluidics
Abstract : We describe how combining microfluidics with TIRF and epifluorescence microscopy can greatly facilitate the quantitative analysis of actin assembly dynamics and its regulation, as well as the exploration of issues that were often out of reach with standard single-filament microscopy, such as the kinetics of processes linked to actin self-assembly or the kinetics of interaction with regulators. We also show how the viscous drag force exerted by fluid flowing on the filaments can be calibrated in order to assess the mechanosensitivity of end-binding protein machineries such as formins or adhesion proteins. We also discuss how microfluidics, in conjunction with other techniques, could be used to address the mechanism of coordination between heterogeneous populations of filaments, or the behavior of individual filaments during regulated treadmilling. These techniques also can be applied to study the assembly and regulation of other cytoskeletal polymers such as microtubules, septins, intermediate filaments, as well as the transport of cargoes by molecular motors under a flow-produced load.
Spire and Formin 2 Synergize and Antagonize in Regulating Actin Assembly in Meiosis by a Ping-Pong Mechanism [PDF LINK] Montaville P, Jégou A, Pernier J, Compper C, Guichard B, Mogessie B, Schuh M, Romet-Lemonne G, Carlier MFPLoS Biol. 2014 ; 12(2): e1001795.
Spire and Formin 2 Synergize and Antagonize in Regulating Actin Assembly in Meiosis by a Ping-Pong Mechanism [PDF LINK]
Abstract : In mammalian oocytes, three actin binding proteins, Formin 2 (Fmn2), Spire, and profilin, synergistically organize a dynamic cytoplasmic actin meshwork that mediates translocation of the spindle toward the cortex and is required for successful fertilization. Here we characterize Fmn2 and elucidate the molecular mechanism for this synergy, using bulk solution and individual filament kinetic measurements of actin assembly dynamics. We show that by capping filament barbed ends, Spire recruits Fmn2 and facilitates its association with barbed ends, followed by rapid processive assembly and release of Spire. In the presence of actin, profilin, Spire, and Fmn2, filaments display alternating phases of rapid processive assembly and arrested growth, driven by a "ping-pong" mechanism, in which Spire and Fmn2 alternately kick off each other from the barbed ends. The results are validated by the effects of injection of Spire, Fmn2, and their interacting moieties in mouse oocytes. This original mechanism of regulation of a Rho-GTPase-independent formin, recruited by Spire at Rab11a-positive vesicles, supports a model for modulation of a dynamic actin-vesicle meshwork in the oocyte at the origin of asymmetric positioning of the meiotic spindle.
Mechanotransduction down to individual actin filaments Romet-Lemonne G, Jégou AEur J Cell Biol. 2013 ; 92(10-11):333-8. - Review
Mechanotransduction down to individual actin filaments
Abstract : The actin cytoskeleton plays an essential role in a cell's ability to generate and sense forces, both internally and in interaction with the outside world. The transduction of mechanical cues into biochemical reactions in cells, in particular, is a multi-scale process which requires a variety of approaches to be understood. This review focuses on understanding how mechanical stress applied to an actin filament can affect its assembly dynamics. Today, experiments addressing this issue at the scale of individual actin filaments are emerging and bring novel insight into mechanotransduction. For instance, recent data show that actin filaments can act as mechanosensors, as an applied tension or curvature alters their conformation and their affinity for regulatory proteins. Filaments can also transmit mechanical tension to other proteins, which consequently change the way they interact with the filaments to regulate their assembly. These results provide evidence for mechanotransduction at the scale of individual filaments, showing that forces participate in the regulation of filament assembly and organization. They bring insight into the elementary events coupling mechanics and biochemistry in cells. The experiments presented here are linked to recent technical developments, and certainly announce the advent of more exciting results in the future.
Dimeric WH2 domains in Vibrio VopF promote actin filament barbed-end uncapping and assisted elongation Pernier J, Orban J, Avvaru BS, Jégou A, Romet-Lemonne G, Guichard B, Carlier MFNat Struct Mol Biol. 2013 ; 20(9):1069-76.
Dimeric WH2 domains in Vibrio VopF promote actin filament barbed-end uncapping and assisted elongation
Abstract : Proteins containing repeats of the WASP homology 2 (WH2) actin-binding module are multifunctional regulators of actin nucleation and assembly. The bacterial effector VopF in Vibrio cholerae, like VopL in Vibrio parahaemolyticus, is a unique homodimer of three WH2 motifs linked by a C-terminal dimerization domain. We show that only the first and third WH2 domains of VopF bind G-actin in a non-nucleating, sequestered conformation. Moreover, dimeric WH2 domains in VopF give rise to unprecedented regulation of actin assembly. Specifically, two WH2 domains on opposite protomers of VopF direct filament assembly from actin or profilin-actin by binding terminal subunits and uncapping capping protein from barbed ends by a new mechanism. Thus, VopF does not nucleate filaments by capping a pointed-end F-actin hexamer. These properties may contribute to VopF pathogenicity, and they show how dimeric WH2 peptides may mediate processive filament growth.
On phosphate release in actin filaments Jégou A, Niedermayer T, Lipowsky R, Carlier MF, Romet-Lemonne GBiophys J. 2013 ; 104(12):2778-9. Comment.
On phosphate release in actin filaments
Comments to the editor on phosphate release in actin filaments
Formin mDia1 senses and generates mechanical forces on actin filaments Jégou A, Carlier MF, Romet-Lemonne GNature Communications 2013 ; 4:1883
Formin mDia1 senses and generates mechanical forces on actin filaments
Abstract : Cytoskeleton assembly is instrumental in the regulation of biological functions by physical forces. In a number of key cellular processes, actin filaments elongated by formins such as mDia are subject to mechanical tension, yet how mechanical forces modulate the assembly of actin filaments is an open question. Here, using the viscous drag of a microfluidic flow, we apply calibrated piconewton pulling forces to individual actin filaments that are being elongated at their barbed end by surface-anchored mDia1 proteins. We show that mDia1 is mechanosensitive and that the elongation rate of filaments is increased up to two-fold by the application of a pulling force. We also show that mDia1 is able to track a depolymerizing barbed end in spite of an opposing pulling force, which means that mDia1 can efficiently put actin filaments under mechanical tension. Our findings suggest that formin function in cells is tightly coupled to the mechanical activity of other machineries.
Mycolactone activation of Wiskott-Aldrich syndrome proteins underpins Buruli ulcer formation [PDF LINK] Guenin-Macé L, Veyron-Churlet R, Thoulouze MI, Romet-Lemonne G, Hong H, Leadlay PF, Danckaert A, Ruf MT, Mostowy S, Zurzolo C, Bousso P, Chrétien F, Carlier MF, Demangel CJ Clin Invest. 2013 ; 123(4):1501-12
Mycolactone activation of Wiskott-Aldrich syndrome proteins underpins Buruli ulcer formation [PDF LINK]
Abstract : Mycolactone is a diffusible lipid secreted by the human pathogen Mycobacterium ulcerans, which induces the formation of open skin lesions referred to as Buruli ulcers. Here, we show that mycolactone operates by hijacking the Wiskott-Aldrich syndrome protein (WASP) family of actin-nucleating factors. By disrupting WASP autoinhibition, mycolactone leads to uncontrolled activation of ARP2/3-mediated assembly of actin in the cytoplasm. In epithelial cells, mycolactone-induced stimulation of ARP2/3 concentrated in the perinuclear region, resulting in defective cell adhesion and directional migration. In vivo injection of mycolactone into mouse ears consistently altered the junctional organization and stratification of keratinocytes, leading to epidermal thinning, followed by rupture. This degradation process was efficiently suppressed by coadministration of the N-WASP inhibitor wiskostatin. These results elucidate the molecular basis of mycolactone activity and provide a mechanism for Buruli ulcer pathogenesis. Our findings should allow for the rationale design of competitive inhibitors of mycolactone binding to N-WASP, with anti-Buruli ulcer therapeutic potential.
Intermittent depolymerization of actin filaments is caused by photo-induced dimerization of actin protomers [PDF LINK] Niedermayer T#, Jégou A#, Chièze L, Guichard B, Helfer E, Romet-Lemonne G*, Carlier MF, Lipowsky R*PNAS 2012 ; 109(27):10769-74
Intermittent depolymerization of actin filaments is caused by photo-induced dimerization of actin protomers [PDF LINK]
Abstract : Actin, one of the most abundant proteins within eukaryotic cells, assembles into long filaments that form intricate cytoskeletal networks and are continuously remodelled via cycles of actin polymerization and depolymerization. These cycles are driven by ATP hydrolysis, a process that also acts to destabilize the filaments as they grow older. Recently, abrupt dynamical changes during the depolymerization of single filaments have been observed and seemed to imply that old filaments are more stable than young ones [Kueh HY, et al. (2008) Proc Natl Acad Sci USA 105:16531–16536]. Using improved experimental setups and quantitative theoretical analysis, we show that these abrupt changes represent actual pauses in depolymerization, unexpectedly caused by the photo-induced formation of actin dimers within the filaments. The stochastic dimerization process is triggered by random transitions of single, fluorescently labeled protomers. Each pause represents the delayed dissociation of a single actin dimer, and the statistics of these single molecule events can be determined by optical microscopy. Unlabeled actin filaments do not exhibit pauses in depolymerization, which implies that, in vivo, older filaments become destabilized by ATP hydrolysis, unless this aging effect is overcompensated by actin-binding proteins. The latter antagonism can now be systematically studied for single filaments using our combined experimental and theoretical method. Furthermore, the dimerization process discovered here provides a molecular switch, by which one can control the length of actin filaments via changes in illumination. This process could also be used to locally “freeze” the dynamics within networks of filaments.
Supramolecular assemblies of lipid-coated polyelectrolytes Tresset G, Lansac Y, Romet-Lemonne GLangmuir. 2012 ; 28(13):5743-52
Supramolecular assemblies of lipid-coated polyelectrolytes
Abstract : We reveal the existence of a general class of supramolecular assemblies made up of lipid-coated polyelectrolytes including the celebrated lipid–nucleic acid complexes. With the aid of high-resolution cryo-electron microscopy, we unveil the nanoscale internal organization of assemblies generated with a wide range of synthetic and biological polyelectrolytes, several of them being investigated in this context for the first time, namely, poly(styrene sulfonic acid), carboxylmethylcellulose, and filamentous actin. Using an original coarse-grained model representing lipid-coated polyelectrolytes as semiflexible tubes, we thoroughly explored the morphologies resulting from the self-assembly process as a function of tube lengths and rigidities; the computed structures are fully consistent with the experimental observations. In particular, we found a strong extension of the correlation range of the order parameter as the rigidity of the lipid-coated polyelectrolytes increases. Electrostatic interactions provide a stabilizing mechanism leading to finite-size equilibrium assemblies. These assemblies may constitute a generic route for interfacing polyelectrolytes to living cells to perform gene delivery, for instance.
Microfluidics pushes forward microscopy analysis of actin dynamics [PDF LINK] Jégou A, Carlier MF, Romet-Lemonne GBioarchitecture. 2011 ; 1(6):271-276
Microfluidics pushes forward microscopy analysis of actin dynamics [PDF LINK]
Abstract : Actin filaments, an essential part of the cytoskeleton, drive various cell processes, during which they elongate, disassemble and form different architectures. Over the past 30 years, the study of actin dynamics has relied mainly on bulk solution measurements, which revealed the kinetics and thermodynamics of actin self-assembly at barbed and pointed ends, its control by ATP hydrolysis and its regulation by proteins binding either monomeric actin or filament ends and sides. These measurements provide quantitative information on the averaged behavior of a homogeneous population of filaments. They have been complemented by light microscopy observations of stabilized individual filaments, providing information inaccessible using averaging methods, such as mechanical properties or length distributions. In the past ten years, the improvement of light microscopy techniques has allowed biophysicists to monitor the dynamics of individual actin filaments, thus giving access to the length fluctuations of filaments or the mechanism of processive assembly by formins. Recently, in order to solve some of the problems linked to these observations, such as the need to immobilize filaments on a coverslip, we have used microfluidics as a tool to improve the observation, manipulation and analysis of individual actin filaments. This microfluidic method allowed us to rapidly switch filaments from polymerizing to depolymerizing conditions, and derive the molecular mechanism of ATP hydrolysis on a single filament from the kinetic analysis of its nucleotide-dependent disassembly rate. Here, we discuss how this work sets the basis for future experiments on actin dynamics, and briefly outline promising developments of this technique.
Jégou A, Niedermayer T, Orbán J, Didry D, Lipowsky R, Carlier MF, Romet-Lemonne GPLoS Biol. 2011 ; 9(9): e1001161
Abstract : The hydrolysis of ATP associated with actin and profilin-actin polymerization is pivotal in cell motility. It is at the origin of treadmilling of actin filaments and controls their dynamics and mechanical properties, as well as their interactions with regulatory proteins. The slow release of inorganic phosphate (Pi) that follows rapid cleavage of ATP gamma phosphate is linked to an increase in the rate of filament disassembly. The mechanism of Pi release in actin filaments has remained elusive for over 20 years. Here, we developed a microfluidic setup to accurately monitor the depolymerization of individual filaments and determine their local ADP-Pi content. We demonstrate that Pi release in the filament is not a vectorial but a random process with a half-time of 102 seconds, irrespective of whether the filament is assembled from actin or profilin-actin. Pi release from the depolymerizing barbed end is faster (half-time of 0.39 seconds) and further accelerated by profilin. Profilin accelerates the depolymerization of both ADP- and ADP-Pi-F-actin. Altogether, our data show that during elongation from profilin-actin, the dissociation of profilin from the growing barbed end is not coupled to Pi release or to ATP cleavage on the terminal subunit. These results emphasize the potential of microfluidics in elucidating actin regulation at the scale of individual filaments.