Biology subjects

Guichard, B.

Publications and source records attributed to Guichard, B..

6 recordsLinked to original sources

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↗

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

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↗

bioRxiv2019

Distribution and abundance of bottlenose dolphin (Tursiops truncatus) over the French Mediterranean continental shelf

The Mediterranean bottlenose dolphin (Tursiops truncatus) sub-population is listed as vulnerable by the International Union for Conservation of Nature. This species is strictly protected in France and the designation of Special Areas of Conservation (SAC) is required under the European Habitat Directive. However, little information is available about the structure, dynamic and distribution of the population in the French Mediterranean waters. We collected photo-identification data over the whole French Mediterranean continental shelf all year round between 2013 and 2015. We sighted 151 groups of bottlenose dolphins allowing the individually photo-identification of 1,060 animals. The encounter rate distribution showed the presence of bottlenose dolphins over the whole continental shelf all year round. Using capture-recapture methods, we estimated for the first time the size of the bottlenose dolphin resident population at 557 individuals (95% confidence interval: 216-872) along the French Mediterranean continental coast. Our results were used in support of the designation of a new dedicated SAC in the Gulf of Lion and provide a reference state for the bottlenose dolphin monitoring in the French Mediterranean waters in the context of the Marine Strategy Framework Directive.

ecology↗

bioRxiv2019

Geometrical constraints greatly hinder formin mDia1 activity

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 crosslinking 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 rates and processivity. When filaments are not bundled, formins can be anchored to static or fluid surfaces, by either end of the proteins, without affecting their activity. We show that filament bundling by fascin reduces both unanchored formin elongation rate and 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 freedoms. 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.

biophysics↗

bioRxiv2017

Modulation of formin processivity by profilin and mechanical tension

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 formin mDia1 dissociates 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 and its confrontation to 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.

biophysics↗