Marie-France Carlier

French National Centre for Scientific Research | CNRS · LEBS

Movie S4. Concentration-Dependent Enhancement of Filament Severing by ADF, Related to Figure 1E Montage of time-lapsed images of effect of ADF filament severing. Filaments were grown from spectrin-actin seeds anchored on the coverslip to a length of about 10μm. The filaments were then aged to ADP-F-actin by exposing to F-buffer for 15 minutes in p...

Movie S1. Enhanced Pointed-End Filament Depolymerization by 0.1 μM ADF, Related to Figure 1C Filaments were grown from coverslip-anchored gelsolin-actin complexes. The barbed ends were bound to anchored Gelsolin and the pointed ends were free in the flow. The filaments were then aged to ADP-F-actin by exposing to F-buffer for 15 minutes. Free poin...

Montage of two separate filaments from the flow (0.75 μM ADF) which incidentally got stuck to the coverslip surface. These filaments with both their ends free, are seen to treadmill with equivalent elongation of their barbed ends (denoted by “+”) and shrinkage of their pointed ends. Constant filament length is maintained during treadmilling. Filame...

Movie S2. Enhanced Pointed-End Filament Depolymerization by 2.5 μM ADF, Related to Figure 1C Filaments were grown from coverslip-anchored gelsolin-actin complexes. The barbed ends were bound to anchored Gelsolin and the pointed ends were free in the flow. The filaments were then aged to ADP-F-actin by exposing to F-buffer for 15 minutes. Free poin...

Movie S3. Concentration-Dependent Enhancement of Filament Barbed-End Depolymerization by ADF, Related to Figure 1D Filaments were grown from coverslip-anchored spectrin-actin seeds with filament barbed ends free in the flow. The filaments were then aged to ADP-F-actin by exposing to F-buffer for 15 minutes. Free barbed ends of these filaments were...

A living cell’s ability to assemble actin filaments in intracellular motile processes is directly dependent on the availability of polymerizable actin monomers, which feed polarized filament growth [1, 2]. Continued generation of the monomer pool by filament disassembly is therefore crucial. Disassemblers like actin depolymerizing factor (ADF)/cofi...

Migration frequently involves Rac-mediated protrusion of lamellipodia, formed by Arp2/3 complex-dependent branching thought to be crucial for force generation and stability of these networks. The formins FMNL2 and FMNL3 are Cdc42 effectors targeting to the lamellipodium tip and shown here to nucleate and elongate actin filaments with complementary...

Cell migration and cell-cell communication involves the protrusion of actin-rich cell surface projections such as lamellipodia and filopodia. Lamellipodia are networks of actin filaments generated and turned over by filament branching through Arp2/3 complex. Inhibition of branching is commonly agreed to eliminate formation and maintenance of lamell...

Supplementary Figures and Supplementary References

Analysis of formin-mediated filament elongation in the presence of profilin.Related to Fig. 1e. Polymerization of 1 μM actin (10% ATTO488- and 5% ATTO565-labelled) in the presence of 5 μM profilin (PFN) and 1 nM of formin fragments as indicated and visualized by TIRF1-M. Top left panel shows growth of control filaments in presence of 5 μM PFN. Fila...

Comparison of lamellipodium protrusion in control and FMNL2/3 knockdown cells.Related to Fig. 2. Phase contrast time-lapse microscopy performed on representative examples of mock RNAi- and FMNL2+3 RNAi-treated B16-F1 cells. Knockdown of FMNL2 and -3 (right panel) reduces average rate of lamellipodium protrusion. In addition, protrusion of lamellipo...

FMNL2/3 knockdown cells exhibit irregular, fluctuating protrusion of lamellipodia.Related to Supplementary Fig. 6c, d: Representative example of time-lapse fluorescence microscopy of lamellipodia formed by mock versus FMNL2/3 RNAi-treated cells expressing EGFP-lifeact. Note that the FMNL2/3 knockdown cell exhibits a narrowed, less efficiently protr...

FMNL3-EGFP is active and localizes to protruding lamellipodia.Related to Fig.1a. Time-lapse fluorescence and phase-contrast microscopy of B16-F1 cell transiently transfected with FMNL3-EGFP, which based on previous experiments with FMNL2 is expected to be myristoylated and thus fully regulated. Consistently, FMNL3-EGFP showed prominent accumulation...

Visualization of FMNL-mediated actin filament generation by TIRF microscopy.Related to Fig. 1b. Polymerization of 1 μM actin (10% ATTO488- and 5% ATTO565-labelled), visualized by TIRF-M alone or upon addition of 10 or 100 nM FMNL2-21P or FMNL3, as indicated. Note that filaments capped by formins grow considerably slower than control filaments, as k...

Actin network assembly rates in FMNL2/3 knockdown versus control cells as determined by FRAP.Related to Supplementary Fig. 8a: Mock RNAi- or FMNL2/3 RNAi-treated cells transiently expressing EGFP-actin were subjected to fluorescence recovery after photobleaching (FRAP) experiments. Due to exclusive incorporation of actin fluorescence from the front...

A living cell’s ability to assemble actin filaments in intracellular motile processes is directly dependent on the availability of polymerizable actin monomers which feed polarized filament growth. Continued generation of the monomer pool by filament disassembly is therefore crucial. Disassemblers like ADF/cofilin and filament cappers like Capping...

Various cellular processes (including cell motility) are driven by the regulated, polarized assembly of actin filaments into distinct force-producing arrays of defined size and architecture. Branched, linear, contractile and cytosolic arrays coexist in vivo, and cells intricately control the number, length and assembly rate of filaments in these ar...

Cell motility and actin homeostasis depend on the control of polarized growth of actin filaments. Profilin, an abundant regulator of actin dynamics, supports filament assembly at barbed ends by binding G-actin. Here, we demonstrate how, by binding and destabilizing filament barbed ends at physiological concentrations, profilin also controls motilit...

Movie S6. Effect of Filament Anchoring on Barbed End Branching, Related to Figure 6 and S5

Movie S4. Effect of Barbed End Interacting Proteins on Filament Branching, Related to Figure 7

Movie S5. VASP Competes with Filament Branching at Barbed Ends, Related to Figure 7

Polarized assembly of actin filaments forms the basis of actin-based motility and is regulated both spatially and temporally. Cells use a variety of mechanisms by which intrinsically slower processes are accelerated, and faster ones decelerated, to match rates observed in vivo. Here we discuss how kinetic studies of individual reactions and cycles...

The Arp2/3 complex consists of seven evolutionarily conserved subunits (Arp2, Arp3 and ARPC1-5) and plays an essential role in generating branched actin filament networks during many different cellular processes. In mammals, however, the ARPC1 and ARPC5 subunits are each encoded by two isoforms that are 67% identical. This raises the possibility th...

Formin 2 (Fmn2), a member of the FMN family of formins, plays an important role in early development. This formin cooperates with profilin and Spire, a WH2 repeat protein, to stimulate assembly of a dynamic cytoplasmic actin meshwork that facilitates translocation of the meiotic spindle in asymmetric division of mouse oocytes. The KIND domain of Sp...

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 t...

Supplementary Figures 1-15

Single molecule imaging of fluorescently labelled formin forming a ternary complex with CP at the barbed end. (Complementary to Figure 1c)

Anchored formins can renucleate new filaments after dissociation of filaments due to CP binding.

Actin cytoskeleton remodeling, which drives changes in cell shape and motility, is orchestrated by a coordinated control of polarized assembly of actin filaments. Signal responsive, membrane-bound protein machineries initiate and regulate polarized growth of actin filaments by mediating transient links with their barbed ends, which elongate from po...

Actin is a major actor in the determination of cell shape. On the one hand, site-directed assembly/disassembly cycles of actin filaments drive protrusive force leading to lamellipodia and filopodia dynamics. Force produced by actin similarly contributes in membrane scission in endocytosis or Golgi remodeling. On the other hand, cellular processes l...

Significance How the continuous polymerization of actin filaments pushes the cell membrane forward in migrating cells is an intriguing issue that has yet to be fully understood. Molecular mechanisms were proposed, but testing them experimentally is a challenge, due to hardly accessible length scales and to the influence of regulatory proteins const...

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 o...

Cordon-Bleu (Cobl) is a regulator of actin dynamics in neural development and ciliogenesis. Its function is associated with three adjacent actin binding WASP Homology 2 (WH2) domains. We showed that these WH2 repeats confer multifunctional regulation of actin dynamics, which makes Cobl a « dynamizer » of actin assembly, inducing fast turnover of ac...

Author Summary Mammalian reproduction requires successful meiosis, which consists of two strongly asymmetric cell divisions. In meiosis I, movement of the spindle (the subcellular structure that segregates chromosomes during division) toward the oocyte cortex (the outer layer of the egg) is essential for fertility. This process requires that actin...

Several pathogens induce propulsive actin comet tails in cells they invade to disseminate their infection. They achieve this by recruiting factors for actin nucleation, the Arp2/3 complex, and polymerization regulators from the host cytoplasm. Owing to limited information on the structural organization of actin comets and in particular the spatial...

WH2 domains are multifunctional regulators of actin assembly that can either sequester G-actin or allow polarized barbed end growth. They all bind similarly to a hydrophobic pocket at the barbed face of actin. Depending on their electrostatic environment, WH2 domains can nucleate actin assembly by facilitating the formation of pre-nuclei dimers alo...

Filopodia explore the environment, sensing soluble and mechanical cues during directional motility and tissue morphogenesis. How filopodia are initiated and spatially restricted to specific sites on the plasma membrane is still unclear. Here, we show that the membrane deforming and curvature sensing IRSp53 (Insulin Receptor Substrate of 53 kDa) pro...

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 thi...

Cellular protrusions involved in motile processes are driven by site-directed assembly of actin filaments in response to Rho-GTPase signalling. So far, only chemical compounds depolymerizing actin or stabilizing filaments, inhibiting N-WASP, Arp2/3 or formins, have been used to eliminate the formation of protrusions, while Rho-GTPase-dominant posit...

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 micr...

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 le...

The study of actin assembly dynamics at the scale of individual filaments provides key information on the molecular mechanisms at play. using microfluidics, we have developed experimental configurations which provide a straightforward and accurate monitoring of individual filaments in vitro, with an extensive control of their biochemical environmen...

The dynamic polymerization (assembly) and depolymerization (disassembly) of actin filaments are pivotal for cell motility, cell adhesion, and cell division. The hydrolysis of actin-bound ATP destabilizes the filaments over time by enhancing the depolymerization velocity. However, abrupt changes of depolymerization were observed in single filament e...

Wiskott-Aldrich syndrome proteins (WASP) are a family of proteins that all catalyze actin filament branching with the Arp2/3 complex in a variety of actin-based motile processes. The constitutively active C-terminal domain, called VCA, harbors one or more WASP homology 2 (WH2) domains that bind G-actin, whereas the CA extension binds the Arp2/3 com...

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. Recent...

The WASP-homology 2 (WH2) domain is a 5-kDa actin-binding protein module that attracts increasing interest by its multifunctional regulation of actin dynamics in motile and morphogenetic processes. Identified by a short consensus sequence LKKT/V originally found in the actin-sequestering ß-thymosin peptides, the ßT/WH2 domains are inserted in a lar...

Toxoplasma gondii belongs to the phylum Apicomplexa, a group of obligate intracellular parasites that rely on gliding motility to enter host cells. Drugs interfering with the actin cytoskeleton block parasite motility, host cell invasion, and egress from infected cells. Myosin A, profilin, formin 1, formin 2, and actin-depolymerizing factor have al...

β-Thymosin (βT) and WH2 domains are widespread, intrinsically disordered actin-binding peptides that display significant sequence variability and different regulations of actin self-assembly in motile and morphogenetic processes. Here, we reveal the structural mechanisms by which, in their 1:1 stoichiometric complexes with actin, they either inhibi...

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 point...

IQGAP1 is a large modular protein that displays multiple partnership and is thought to act as a scaffold in coupling cell signaling to the actin and microtubule cytoskeletons in cell migration, adhesion, and cytokinesis. However the molecular mechanisms underlying the activities of IQGAP1 are poorly understood in part because of its large size, poo...

Comparison of depolymerization traces from different individual filaments, under different conditions. Filaments were depolymerized in F-buffer after elongation from (A) MgATP-actin or (B) MgADP-actin. Filaments were depolymerized (C) in the presence of 20 µM profilin after elongation from MgATP-actin, or (D) in the presence of 40 µM profilin after...

Pauses occurring during depolymerization are unrelated to the acceleration of depolymerization, which reflects the ADP-Pi content of the filament. Left: Length versus time for a filament depolymerizing with 80 µM profilin. Depolymerization is interrupted by a pause between 100 and 150 s after the beginning of depolymerization. Right: 1/vdepol versu...

Depolymerization of an ADP-actin filament obtained by aging. A filament grown with 2 µM MgATP-actin is then left to age at constant length in the presence of 0.1 µM actin (steady-state concentration for the barbed end) for 6 min, before initiating depolymerization at time t = 0. (PDF)

Direct fit of depolymerization curve to experimental data. The theoretical curve, given by the differential equation 12 of the supporting text, is fitted to the depolymerization curves of six filaments from one experiment. These curves were slightly shifted in a vertical direction to have a common initial length. Inset: The theoretical curve is fit...

The depolymerization of ADP-actin filaments slows down upon exposure to Pi. A filament elongated from MgADP-actin was depolymerized in standard F-buffer for 60 s (blue diamonds) then in the presence of 25 mM Pi (red squares). Lines represent linear fits of the data. At this resolution, the transition to a slow depolymerization rate appears instanta...

The effect of profilin during depolymerization is fully reversible. A depolymerizing filament is exposed to 100 µM profilin for 1 min and subsequently switched back to depolymerization in buffer without profilin. (PDF)

Comparison of simulations with analytical results. We simulated the polymerization and depolymerization of 10,000 filaments with rates as specified in Text S1, i.e. including ATP cleavage. Average values (blue dots) ± standard deviations (blue dashed lines) are depicted. The continuous red line is the solution of equation 12 of Text S1 for the same...

Filaments do not elongate from MgADP-G-actin in the presence of profilin. (A) Barbed end and pointed end growths from 16.4 µM (blue) and 10.5 µM (red) MgADP-G-actin (2% pyrene-labeled) were initiated using 0.2 nM spectrin-actin seeds (open circles) or 10 nM gelsolin-actin seeds (closed squares), respectively, in the presence of the indicated amount...

(I) Additional information on the pauses that occur during depolymerization. (II) Theoretical analysis for the depolymerizaton of actin filaments, (III) analysis of length-versus-time data for depolymerizing filaments, (IV) stochastic simulations. (PDF)

The length of the ADP-Pi F-actin at growing barbed ends depends on the age of filaments. Filaments growing in coherent fashion in the presence of 3 µM G-actin (50% pyrene-labeled) and the indicated amounts of spectrin-actin seeds (s.a.s.) were depolymerized by 6-fold dilution in F buffer in the presence of 5 µM Latrunculin A as soon as 20% of actin...

Individual actin filament, elongated and subsequently depolymerized in a microfluidic flow. The filament is elongated for 6 min, from an initial length of 2.4 µm to a final length of 14.3 µm (30 s between images, acquired in TIRF microscopy). It is depolymerized by switching to buffer with no actin, and depolymerization lasts 12 min (20 s between i...

Simulation of the length during polymerization and depolymerization. The rates are specified in the Text S1. Top: A cap of ATP-actin is present during polymerization, but not during depolymerization. Number of ATP-subunits (red), ADP-Pi-subunits (black), ADP-subunits (green), and overall number (blue). Bottom: Fluctuations indicated by 20 randomly...

One of the most versatile regulators of actin assembly, the WASP homology 2 (WH2) domain, reveals previously unknown facets by combining with a newly discovered actin-nucleating dimeric structure in the effector protein VopL from Vibrio parahaemolyticus.

Author Summary Actin proteins assemble into microfilaments that control cell shape and movement by polymerizing or depolymerizing. These actin monomers can bind ATP or ADP molecules. The incorporation of an ATP-actin monomer into a growing filament results in rapid cleavage of ATP into ADP and inorganic phosphate (Pi), followed by a slower release...

Cordon-Bleu is, like Spire, a member of the growing family of WH2 repeat proteins, which emerge as versatile regulators of actin dynamics. They are expressed in morphogenetic and patterning processes and nucleate actin assembly in vitro. Here, we show that Cordon-Bleu works as a dynamizer of actin assembly by combining many properties of profilin w...

IQGAP1 is a large modular protein that displays multiple partnership and is thought to act as a scaffold in coupling cell signaling to the actin and microtubule cytoskeletons in cell migration, adhesion, and cytokinesis. However the molecular mechanisms underlying the activities of IQGAP1 are poorly understood in part because of its large size, poo...

Magnetic colloids move apart as a consequence of actin polymerization. Video of the magnetic chain during actin polymerization. (9.52 MB AVI)