2017 :

ADF/Cofilin Accelerates Actin Dynamics by Severing Filaments and Promoting Their Depolymerization at Both Ends
Hugo Wioland, Berengere Guichard, Yosuke Senju, Sarah Myram, Pekka Lappalainen, Antoine Jegou & Guillaume Romet-Lemonne
Current Biology 2017 130: 1509-1517 [PDF LINK]

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
Stéphane Frémont, Guillaume Romet-Lemonne, Anne Houdusse & Arnaud Echard
Journal of Cell Science 2017 130: 1509-1517 [PDF LINK]
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

Stéphane Frémont, Hussein Hammich, Jian Bai, Hugo Wioland, Kerstin Klinkert, Murielle Rocancourt, Carlos Kikuti, David Stroebel, Guillaume Romet-Lemonne, Olena Pylypenko, Anne Houdusse & Arnaud Echard
Nature Communications 2017 8:14528  [PDF LINK]

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.

2016 :

Single Filaments to Reveal the Multiple Flavors of Actin
Antoine Jégou & Guillaume Romet-Lemonne
Biophysical Journal 110(10) 2238  [PDF LINK]

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.

2015 :

Formin and capping protein together embrace the actin filament in a ménage à trois
Shashank Shekhar, Mikael Kerleau, Sonja Kühn, Julien Pernier, Guillaume Romet-Lemonne, Antoine Jégou & Marie-France Carlier
Nature Communications 2015 4:8730 [PDF LINK]

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.

2014 :

Cellular control of cortical actin nucleation

Bovelaan 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 G

Current Biology 2014 ; 24(14): 1628-35.


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 [4], and tissue morphogenesis [5]. For example, cortex contractility plays a crucial role in amoeboid migration of metastatic cells [6] and during division, where its misregulation can lead to aneuploidy [7]. 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 A.
Methods Enzymol. 2014 ; 540:3-17.


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.

Montaville P, Jégou A, Pernier J, Compper C, Guichard B, Mogessie B, Schuh M, Romet- Lemonne G, Carlier MF.
PLoS Biol. 2014 ; 12(2): e1001795. [PDF LINK]


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.

2013 :

Mechanotransduction down to individual actin filaments.
Romet-Lemonne G, Jégou A.
Eur J Cell Biol. 2013 ; 92(10-11):333-8. Review.


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 MF.
Nat Struct Mol Biol. 2013 ; 20(9):1069-76.


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 G.
Biophys J. 2013 ; 104(12):2778-9. Comment.

Formin mDia1 senses and generates mechanical forces on actin filaments.
Jégou A, Carlier MF, Romet-Lemonne G.
Nature Communications 2013 ; 4:1883.

 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.
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 C.
J Clin Invest. 2013 ; 123(4):1501-12 [PDF LINK]

2012 :

Intermittent depolymerization of actin filaments is caused by photo-induced  dimerization of actin protomers.

Niedermayer T, Jégou A, Chièze L, Guichard B, Helfer E, Romet-Lemonne G, Carlier MF,  Lipowsky R.

PNAS 2012 ; 109(27):10769-74. [PDF LINK]
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 G.
Langmuir. 2012 ; 28(13):5743-52.

2011 :

Microfluidics pushes forward microscopy analysis of actin dynamics.
Jégou A, Carlier MF, Romet-Lemonne G.
Bioarchitecture. 2011 ; 1(6):271-276. [PDF LINK]

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.

Individual Actin Filaments in a Microfluidic Flow Reveal the Mechanism of ATP  Hydrolysis and Give Insight Into the Properties of Profilin.

Jégou A, Niedermayer T, Orbán J, Didry D, Lipowsky R, Carlier MF, Romet-Lemonne G

PLoS Biol. 2011 ; 9(9): e1001161. [PDF LINK]


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.