Biology subjects

Romet-Lemonne, G.

Publications and source records attributed to Romet-Lemonne, G..

At least 19 recordsLinked to original sources

bioRxiv2026

Tropomodulin and Leiomodin Differentially Nucleate Tropomyosin Assembly on Actin Filaments

Tropomyosins play a central role in the regulation of the actin cytoskeleton by assembling along filaments into rope-like, head-to-tail polymers ("clusters") along filaments that regulate the binding and activity of numerous actin-binding proteins. Importantly, in cells, tropomyosins do not decorate actin filaments uniformly; some filaments are devoid of tropomyosins, whereas others recruit specific isoforms. To explore the diversity of tropomyosin-isoform binding kinetics and identify factors that control their assembly, we designed tropomyosin constructs, bearing a natural N-terminal acetylation and targeted cysteine mutations for fluorescence labelling. We analysed the binding of different tropomyosin isoforms to individual actin filaments reconstituted inside a microfluidic chamber. We show that, despite significant differences in spontaneous nucleation and elongation rates, all tested tropomyosin isoforms can coassemble within the same clusters. In addition, we identified tropomodulin and leiomodin, two pointed-end-binding proteins, to efficiently nucleate the formation of tropomyosin clusters on actin filaments, for all tested tropomyosin isoforms. Notably, while tropomodulin nucleates tropomyosin clusters from the pointed end, leiomodin does so along the entire filament.

biochemistry↗

bioRxiv2026

Action of cofilin on ADP-Pi-containing actin filaments

In cells, the disassembly of actin filaments is tightly controlled by molecular mechanisms where cofilin plays a central role. Cofilin binds to actin filaments cooperatively, forming clusters that lead to the severing of the filaments. Since cofilin binds preferentially filaments that have hydrolysed ATP and released inorganic phosphate (Pi), most studies have focused on the action of cofilin on ADP-actin filaments. Here, we ask how cofilin binds and severs filaments that still contain a large fraction of ADP{middle dot}Pi-actin. To answer this, we use microfluidics and fluorescence microscopy to monitor single filaments exposed to cofilin in controlled conditions, allowing us to disentangle and to quantify the different reactions. We find that the step limiting cluster nucleation is the binding of 2 cofilins on a minimal group of 3-4 contiguous ADP-actin subunits. Cofilin clusters then expand mostly in the direction of the pointed end of the filament, by easily binding to the ADP-actin neighbor of an ADP{middle dot}Pi-actin subunit, whose Pi release it accelerates. Severing at the ADP{middle dot}Pi-actin/cofilin cluster boundary is 30-fold slower than for an ADP-actin boundary. Globally, it seems that cofilin senses and modifies the nucleotide state of the actin subunits that are in its direct vicinity only. Our results provide a more comprehensive understanding of the action of cofilin, taking into account the nucleotide-state complexity of actin filaments.

biochemistry↗

bioRxiv2026

Myosin-X sorts actin filaments into parallel bundles and tunes barbed-end dynamics

Whether a molecular motor can spontaneously organize actin filaments into bundles has remained an open question despite decades of in vitro investigation. Here, we show that the dimeric myosin-X, essential for filopodia initiation and extension in cells, is capable of sorting actin filaments into parallel bundles, gathering barbed ends within 1-2 micrometers. We observe that myosin-X processivity is comparable on single filaments and on bundles induced by myosin-X or fascin. Upon reaching barbed ends, myosin-X slows down the addition or removal of actin subunits, in a myosin density-dependent manner. Furthermore, the funneling of myosin-X towards the remaining filaments at the bundle tip increases motor density and triggers dynamic clustering. Together, we propose that the motor activity of myosin-X is sufficient to initiate filopodia, independently of passive crosslinkers such as fascin or fimbrin.

biochemistry↗

bioRxiv2025

SPIN90 modulates the architecture of lamellipodial actin in an ARPC5L dependent fashion.

When stimulated by nucleation-promoting factors such as WAVE, the Arp2/3 complex generates branched actin networks. In contrast, when activated by SPIN90, the Arp2/3 complex generates linear actin filaments. The Arp2/3 complex in mammals, consists of 8 iso-complexes with dieerent properties as there are two isoforms of Arp3, ArpC1 and ArpC5. Here, using recombinant Arp2/3 iso-complexes with defined compositions, we show that SPIN90 selectively activates ArpC5L- rather than ArpC5- containing complexes to generate linear actin filaments. Consistent with this, SPIN90 at the leading edge of migrating cells enhances the recruitment of ArpC5L-, but not ArpC5- containing, Arp2/3 complexes to lamellipodia. These SPIN90-Arp2/3-ArpC5L complexes generate linear actin filaments that integrate into lamellipodia and impact protrusion eeiciency. Moreover, using polarised light microscopy, we show that loss of SPIN90 leads to an enrichment in actin filaments oriented more perpendicular to the plasma membrane. Our results demonstrate that SPIN90 regulates the architecture and dynamics of lamellipodial actin in an Arp2/3 iso-complex dependent fashion. SummarySPIN90 selectively recruits and activates ArpC5L containing Arp2/3 to shape the architecture and dynamics of lamellipodial actin.

cell biology↗

bioRxiv2025

Rapid aging and disassembly of actin filaments from two evolutionary distant yeasts

Actin is an essential cytoskeletal protein that is extremely well conserved across the tree of life. Similarities and differences in the way actin from different species self-assembles into filaments inform our understanding of its evolution. However, this basic knowledge is largely incomplete. Here, we address this issue by systematically characterizing assembly kinetics for actin from two yeast species that are five hundred million years apart in evolution, Saccharomyces cerevisiae and Schizosaccharomyces pombe, and compare them to the well-studied actin from rabbit skeletal muscle from which they diverged a billion years ago. By monitoring individual actin filaments under controlled conditions, we accurately quantified their barbed end elongation from ATP-actin monomers, their disassembly in the ADP{middle dot}Pi and ADP states, as well as the release of inorganic phosphate, which marks their aging. We find that, in the ATP state, both yeast actins behave strikingly like mammalian actin at filament barbed ends. In contrast, yeast actin filaments in both the ADP{middle dot}Pi and the ADP states depolymerize several-fold faster than their mammalian counterparts, and they release inorganic phosphate over 20-fold faster. We show that the absence of methylation on histidine 73 largely accounts for this faster aging of yeast actin filaments. We also reveal differences between the actins of the two yeasts. In particular, ADP-actin filaments depolymerize faster and are mechanically more flexible in Saccharomyces cerevisiae compared to Schizosaccharomyces pombe. Our findings suggest that actins expressed across species possess more diverse and specialized biochemical characteristics than previously recognized.

biochemistry↗

bioRxiv2025

Vimentin promotes actin assembly by stabilizing ATP-actin subunits at the barbed end

Vimentin intermediate filaments play essential roles in maintaining cell integrity and regulating numerous cellular functions. In particular, vimentin cooperates with the actin cytoskeleton in key cellular processes that rely on actin dynamics, such as migration, division, and mechanosensing. While there is evidence that these two cytoskeletal components interact in cells, the underlying molecular mechanisms are only partially understood. Actin and vimentin can interact through biochemical signaling pathways or via cross-linkers, but whether they engage in a direct protein-protein interaction has remained controversial, in part because such interactions are difficult to isolate and characterize in cells. Using in vitro reconstitution coupled to theoretical modeling, and total internal reflection fluorescence microscopy to monitor the elongation of single actin filaments, we show that vimentin promotes actin elongation by stabilizing actin subunits at the barbed end in a dose-dependent manner. Strikingly, this effect depends on the nucleotide state of actin, as the acceleration is only observed for the elongation from ATP-actin, and not ADP-actin monomers. We further establish that neither the vimentin tail nor head domains are required for this effect, and both filamentous and non-filamentous vimentin enhance actin elongation. Finally, we find that vimentin promotes the nucleation of actin filaments. Consistently, magnetic pull-down assays demonstrate a direct interaction between vimentin and ATP-actin monomers. Altogether, these findings identify vimentin as an unexpected new actor in the regulation of actin dynamics at the barbed end and bring new insights into the functional role of vimentin through cytoskeletal crosstalk.

biophysics↗

bioRxiv2025

Inorganic phosphate in Arp2/3 complex acts as a rapid switch for the stability of actin filament branches

The rate at which actin filaments turn over modulates actin network architecture and mechanosensitivity. Actin filaments often appear as branches, nucleated by the Arp2/3 complex. Within the Arp2/3 complex, Arp2 and Arp3 are ATPases, which, similar to actin, may adopt slightly different conformations depending on their bound nucleotide. We investigate the impact of the nucleotide state of Arp2/3 complexes on branch stability, by mechanically pulling on them. Remarkably, we find that branches with mammalian ADP-Pi and ADP-Arp2/3 complexes detach with the same exponential increase as a function of force, but ADP-Pi-Arp2/3 complex branch junctions are 20 times more stable at all forces. We observe that inorganic phosphate (Pi) is in rapid equilibrium with ADP-Arp2/3 at the branch junction, and is released at a rate greater than 1 s-1. Upon branch dissociation, the surviving Arp2/3 complex, remaining attached to the mother filament, is a thousand times more stable in the ADP-Pi than in the ADP state. Surprisingly, branch regrowth from surviving ADP-Pi-Arp2/3 complex does not require reloading ATP, indicating that the Arp2/3 complex remained in an active state upon debranching. We reveal that GMF destabilizes ADP-Arp2/3 complex branch junctions by accelerating first the dissociation of the daughter filament, and then the dissociation of the surviving Arp2/3 complex from the mother filament. We also report that cortactin stabilizes branches in a force dependent manner, and enhances branch renucleation. GMF and cortactin do not bind to Arp2/3 in the ADP-Pi state. Overall, Our results give a new importance to cytoplasmic Pi, as a possible regulator of branched actin network stability.

biophysics↗

bioRxiv2025

Probing protein-protein interactions with drag flow: A case study of F-actin and tropomyosin

Tropomyosin are central regulators of the actin cytoskeleton, controlling the binding and activity of the other actin binding proteins. The interaction between tropomyosin and actin is quite unique: single tropomyosin dimers bind weakly to actin filaments but get stabilised by end-to-end attachment with neighbouring tropomyosin dimers, forming clusters which wrap around the filament. Force spectroscopy is a powerful approach for studying protein-protein interactions, but classical methods which usually pull with pN forces on a single protein pair, are not well adapted to tropomyosins. Here, we propose a method in which a hydrodynamic drag force is applied directly to the proteins of interest, by imposing a controlled fluid flow inside a microfluidic chamber. The breaking of the protein bonds is directly visualised with fluorescence microscopy. Using this approach, we reveal that very low forces from 0.01 to 0.1 pN per tropomyosin dimer trigger the detachment of entire tropomyosin clusters from actin filaments. We show that the tropomyosin cluster detachment rate depends on the cytoplasmic tropomyosin isoform (Tpm1.6, 1.7, 1.8) and increases exponentially with the applied force. These observations lead us to propose a cluster detachment model which suggests that tropomyosins dynamically explore different positions over the actin filament. Our experimental setup can be used with many other cytoskeletal proteins, and we show, as a proof-of-concept, that the velocity of myosin-X motors is reduced by an opposing fluid flow. Overall, this method expands the range of protein-protein interactions that can be studied by force spectroscopy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=173 HEIGHT=200 SRC="FIGDIR/small/653996v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@19298b4org.highwire.dtl.DTLVardef@16766beorg.highwire.dtl.DTLVardef@18a70c6org.highwire.dtl.DTLVardef@1f1fad5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Arp2/3-mediated bidirectional actin assembly by SPIN90 dimers in metazoans

Branched actin networks nucleated by the Arp2/3 complex play critical roles in various cellular processes, from cell migration to intracellular transport. However, when activated by WISH/DIP/SPIN90 family proteins, Arp2/3 nucleates linear actin filaments. Unexpectedly, we found that human SPIN90 is a dimer that can nucleate bidirectional actin filaments. To understand the basis for this, we determined a 3 [A] resolution structure of human SPIN90-Arp2/3 complex nucleating actin filaments. Our structure shows that SPIN90 dimerises via a 3-helix bundle and interacts with two Arp2/3 complexes. Each SPIN90 molecule binds both Arp2/3 complexes to promote their activation. Our analysis demonstrates that single filament nucleation by Arp2/3 is mechanistically more like branch formation than previously appreciated. The dimerisation domain in SPIN90 orthologues is conserved in metazoans, suggesting that this mode of bidirectional nucleation is a common strategy to generate anti-parallel actin filaments.

biophysics↗

bioRxiv2024

HIV-1 budding requires cortical actin disassembly by the oxidoreductase MICAL1

Many enveloped viruses bud from the plasma membrane that is tightly associated with a dense and thick actin cortex. This actin network represents a significant challenge for membrane deformation and scission, and how it is remodeled during the late steps of the viral cycle is largely unknown. Using super-resolution microscopy, we show that HIV-1 buds in areas of the plasma membrane with low cortical F-actin levels. We find that the cellular oxidoreductase MICAL1 locally depolymerizes actin at budding sites to promote HIV-1 budding and release. In the absence of MICAL1, F-actin abnormally remains at viral budding sites, incompletely budded viruses accumulate at the plasma membrane and viral release is impaired. Remarkably, normal viral release can be restored in MICAL1-depleted cells by inhibiting Arp2/3-dependent branched actin networks. Mechanistically, we find that MICAL1 directly disassembles branched-actin networks and controls the timely recruitment of the ESCRT scission machinery during viral budding. In addition, the MICAL1 activator Rab35 is recruited at budding sites, functions in the same pathway as MICAL1 and is also required for viral release. This work reveals a role for oxidoreduction in triggering local actin depolymerization to control HIV-1 budding, a mechanism that may be widely used by other viruses. The debranching activity of MICAL1 could be involved beyond viral budding in various other cellular functions requiring local plasma membrane deformation.

cell biology↗

bioRxiv2024

Continuous self-repair protects vimentin intermediate filaments from fragmentation

Intermediate filaments are key regulators of cell mechanics. Vimentin, a type of intermediate filament expressed in mesenchymal cells and involved in migration, forms a dense network in the cytoplasm that is constantly remodeling through filament transport, elongation/shortening, and subunit exchange. While it is known that filament elongation involves end-to-end annealing, the reverse process of filament shortening by fragmentation remains unclear. Here, we use a combination of in vitro reconstitution, probed by fluorescence imaging and AFM, with theoretical modeling to uncover the molecular mechanism involved in filament breakage. We first show that vimentin filaments are composed of two populations of subunits, half of which are exchangeable and half immobile. We also show that the exchangeable subunits are tetramers. Furthermore, we reveal a mechanism of continuous filament self-repair, where a soluble pool of vimentin tetramers in equilibrium with the filaments is essential to maintain filament integrity. Filaments break due to local fluctuations in the number of tetramers per cross-section, induced by the constant subunit exchange. We determine that a filament tends to break if approximately four tetramers are removed from the same filament cross-section. Finally, we analyze the dynamics of association/dissociation and fragmentation to estimate the binding energy of a tetramer to a complete versus a partially disassembled filament. Our results provide a comprehensive description of vimentin turnover and reveal the link between subunit exchange and fragmentation. SIGNIFICANCE STATEMENTIntermediate filaments such as vimentin are key contributors to cell mechanics. These filaments are continuously assembled and disassembled by the cell as it grows and moves. While the assembly process is relatively well characterized, here we investigate their much less understood disassembly. We find that the filaments continuously exchange their constitutive proteins with the surrounding solution throughout their length and when too many subunits are removed, the filament breaks. This process sharply differs from the length regulation mechanisms known to occur in other types of filaments involved in the regulation of cell mechanics. Importantly, we also identify two distinct populations of subunits within the filaments, which raises new questions about vimentin intermediate filament structure.

biophysics↗

bioRxiv2023

Regeneration of actin filament branches from the same Arp2/3 complex

Branched actin filaments are found in many key cellular structures. Branches are nucleated by the Arp2/3 complex activated by nucleation-promoting factor (NPF) proteins and bound to the side of pre-existing mother filaments. Over time, branches dissociate from their mother filament, leading to network reorganization and turnover, but this mechanism is less understood. Here, using microfluidics and purified proteins, we examined the dissociation of individual branches under controlled biochemical and mechanical conditions. We observe that Arp2/3 remains bound to the mother filament after most debranching events, even when accelerated by force. Unexpectedly, this mother-remaining Arp2/3 readily nucleates a new actin filament branch, without being activated anew by an NPF: it simply needs to exchange its nucleotide and bind an actin monomer. The protein GMF, which accelerates debranching, prevents branch re-nucleation. Our results suggest that actin filament re-nucleation can provide a self-repair mechanism, helping branched networks to sustain mechanical stress in cells over extended periods of time.

biophysics↗

bioRxiv2023

Fascin-induced bundling protects actin filaments from disassembly by cofilin

Actin filament turnover plays a central role in shaping actin networks, yet the feedback mechanism between network architecture and filament assembly dynamics remains unclear. The activity of ADF/cofilin, the main protein family responsible for filament disassembly, has been mainly studied at the single filament level. Here, we report that fascin, by crosslinking filaments into bundles, strongly slows down filament disassembly by cofilin. We show that this is mainly due to a slower nucleation of the first cofilin clusters, which occurs up to 100-fold slower on large bundles compared to single filaments. In contrast, severing at cofilin cluster boundaries is unaffected by fascin bundling. After the nucleation of an initial cofilin cluster on a filament of a bundle, we observe the local removal of fascin. Surprisingly, the nucleation of cofilin clusters on adjacent filaments is highly enhanced, locally. We propose that this inter-filament cooperativity in cofilin binding arises from the local propagation of the cofilin-induced change in helicity from one filament to the other filaments of the bundle. Taken together, these observations reveal the molecular events explaining why, despite inter-filament cooperativity, fascin crosslinking protects actin filaments from cofilin-induced disassembly. These findings highlight the important role played by crosslinkers in organizing actin networks and modulating the activity of other regulatory proteins.

biochemistry↗

bioRxiv2022

Direct observation of the conformational states of formin mDia1 at actin filament barbed ends and along the filament.

The fine regulation of actin polymerization is essential to control cell motility, architecture and to perform essential cellular functions. Formins are key regulators of actin filament assembly, known to processively elongate filament barbed ends and increase their polymerization rate. Based on indirect observations, different models have been proposed to describe the molecular mechanism governing the processive motion of formin FH2 domains at polymerizing barbed ends. Using electron microscopy, we directly identified two conformations of the mDia1 formin FH2 domains in interaction with the barbed ends of actin filaments. These conformations agree with the open and closed conformations of the "stair stepping" model proposed by Otomo and colleagues1. We observed the FH2 dimers to be in the open conformation for 79% of the data, interacting with the two terminal actin subunits of the barbed end, while they interact with three actin subunits in the closed conformation. Further, our data reveal that the open state encompasses a continuum of states where the orientation of the leading FH2 domain with respect to the filament long axis varies from 108 to 135 degrees. In addition, we identified FH2 domains encircling the core of actin filaments, providing structural information for mDia1 away from the barbed end. Based on these direct observations, we propose a model of formin in interaction with the growing filament end, as well as with the core of the filament.

biophysics↗

bioRxiv2022

Nucleation and stability of branched versus linear Arp2/3-generated actin filaments

Activation of the Arp2/3 complex by VCA-domain-bearing NPFs results in the formation of daughter actin filaments branching off the sides of pre-existing mother filaments. Alternatively, when stimulated by SPIN90, Arp2/3 directly nucleates linear actin filaments. Uncovering the similarities and differences of these two activation mechanisms is fundamental to understanding the regulation and function of Arp2/3. Analysis of individual filaments reveals that the catalytic VCA domain of WASP, N-WASP and WASH, accelerate the Arp2/3-mediated nucleation of linear filaments by SPIN90, in addition to their known branch-promoting activity. Unexpectedly, these VCA domains also destabilize existing branches, as well as SPIN90-Arp2/3 at filament pointed ends. Furthermore, cortactin and GMF, which respectively stabilize and destabilize Arp2/3 at branch junctions, have a similar impact on SPIN90-activated Arp2/3. However, unlike branch junctions, SPIN90-Arp2/3 at the pointed end of linear filaments is not destabilized by piconewton forces, and does not become less stable with time. It thus appears that linear and branched Arp2/3-generated filaments respond similarly to regulatory proteins, albeit with quantitative differences, and that they differ greatly in their responses to aging and to mechanical stress. These results indicate that SPIN90- and VCA-activated Arp2/3 complexes adopt similar yet non-identical conformations, and that their turnover in cells may be regulated differently.

biochemistry↗

bioRxiv2020

Dynamics of Tpm1.8 domains on actin filaments with single moleculeresolution

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.

biophysics↗

bioRxiv2020

The Arp1/11 Minifilament of Dynactin Primes the Endosomal Arp2/3 Complex

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. One Sentence SummaryDendritic actin networks grow in an autocatalytic manner starting from the uncapped minifilament of Dynactin.

cell biology↗

bioRxiv2020

IN VITRO RECONSTITUTION OF DYNEIN FORCE EXERTION IN BULK CYTOPLASM

The forces generated by Microtubules (MTs) and their associated motors orchestrate essential cellular processes ranging from vesicular trafficking to centrosome positioning [1, 2]. 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 [2-4]. 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 [5-14], 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.

biophysics↗