Biology subjects

JEGOU, A.

Publications and source records attributed to JEGOU, A..

11 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

A comprehensive Arabidopsis transcription factor binding atlas reveals pervasive positional and syntactic organization of their DNA binding

Transcription factor (TF) binding underlies gene regulation, yet the determinants of TF-DNA interactions across plant genomes remain incompletely understood. Here, we present a comprehensive atlas of TF binding in Arabidopsis thaliana, generated by reanalyzing 1,157 publicly available ChIP-seq and DAP/ampDAP-seq datasets using a unified processing framework. After stringent curation, 681 high-quality experiments were retained, providing binding information for 425 TFs across 42 families and 23 structural classes. Using this resource, we show that TF binding predictability varies widely across TFs, with family identity explaining substantially more variation in predictive performance than structural class. Among the tested TF binding site (TFBS) prediction models, deep learning approaches achieved the highest overall predictive accuracy, while classical position weight matrices remained competitive and readily interpretable. Beyond motif recognition, we uncover widespread organizational principles of TF binding. TFBS positioning relative to transcription start sites is strongly family-dependent and more concentrated near promoters in vivo. Moreover, preferred spacing between homotypic TFBS is pervasive across TF families, indicating that binding-site syntax is a general organizational feature of TF binding in plants. Comparison of in vivo and in vitro binding profiles further identifies a shared core of sequence-driven binding alongside in vivo-enriched sites associated with non-canonical or composite sequence features. Together, these results highlight the interplay between intrinsic DNA recognition, higher-order binding syntax, and cellular context in shaping TF binding landscapes, while providing a broadly useful resource for the plant community.

plant biology↗

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

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↗

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

Cortactin stabilizes actin branches by bridging activated Arp2/3 to its nucleated actin filament

Regulation of the assembly and turnover of branched actin filament networks nucleated by the Arp2/3 complex is essential during many cellular processes including cell migration and membrane trafficking. Cortactin plays a key role in stabilizing actin filament branches by interacting with the Arp2/3 complex and actin filaments via its N-terminal Acidic domain (NtA) and 6.5 central unstructured 37 amino acid repeats, respectively 1, but the mechanism of this is unclear. We determined the structure of cortactin-stabilized Arp2/3 actin branches using cryo-electron microscopy. We find that cortactin interacts with the new daughter filament nucleated by the Arp2/3 complex at the branch site rather than the initial mother actin filament. Cortactin preferentially binds activated Arp3 in contrast to other nucleation promoting factors (NPFs) 2,3. Cortactin also stabilizes the F-actin-like interface of activated Arp3 with the first actin subunit of the new filament, and its central repeats extend along successive daughter filament subunits. Cortactin binding to Arp3 is incompatible with NPF interaction and its preference for activated Arp3 explains why it is retained at the actin branch. Our data have uncovered why cortactin displaces NPFs, while at the same time promoting synergy to regulate branched actin network dynamics.

biophysics↗

bioRxiv2022

????-actin plasticity is modulated by coordinated actions of histidine 73 methylation, nucleotide type, and ions

The functional importance of the methylation of histidine 73 (H73) in actin remains unclear. Focusing on cytoplasmic {beta}-actin, present in all mammalian cells, we use molecular dynamics simulations with a polarizable force field and adaptive sampling to examine the effects of H73 methylation. Our results show that methylation enhances nucleotide binding cleft opening, alters allosteric pathways connecting subdomains 2 and 4 (SD2 and SD4) in G-actin, and affects backdoor openings and inorganic phosphate release in F-actin, as validated by biochemical assays. These effects depend on the nucleotide and ions interacting with the actin. Together, our findings reveal how H73 methylation regulates {beta}-actin plasticity and integrates environmental cues.

biophysics↗

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↗

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

Actin filament oxidation by MICAL1 suppresses protections from cofilin-induced disassembly

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. Two essential factors normally prevent filament disassembly: the inactivation of cofilin by phosphorylation, and the protection of filaments by tropomyosins, but whether actin oxidation might interfere with these safeguard mechanisms is unknown. 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 severing, in spite of being decorated by tropomyosin, and without requiring the activation of cofilin.

biochemistry↗