Biology subjects

Michelot, A.

Publications and source records attributed to Michelot, A..

11 recordsLinked to original sources

bioRxiv2026

Intrafilament nucleotide exchange in a prokaryotic actin homolog

Polymerization and disassembly govern the cellular functions of cytoskeletal proteins. Canonical nucleotide-dependent polymers, including actin and microtubules, renew their nucleotide state through subunit subunit dissociation, nucleotide exchange in solution and repolymerization. Yet the assembly dynamics of the membrane-bound bacterial actin MreB remain unresolved. Here, using total internal reflection fluorescence microscopy and high-speed atomic force microscopy, we visualise Bacillus subtilis MreB assembly on supported lipid bilayers in real time. ATP binding drives polymerization into symmetrically elongating filaments, whereas ATP hydrolysis within filaments promotes disassembly. Unexpectedly, nucleotides continuously exchange within membrane-bound filaments without detectable subunit turnover, coupling filament stability to the surrounding nucleotide pool: ATP exchange stabilizes filaments, whereas ADP exchange triggers rapid fragmentation and disassembly. Monte-Carlo modelling and single-cell in vivo imaging support this mechanism. Thus, intrafilament nucleotide exchange enables MreB filaments to renew their nucleotide state without polymer turnover, revealing nucleotide-state rejuvenation as a new mode of biological polymer regulation.

microbiology↗

bioRxiv2026

ATP-driven membrane binding and polymerization of bacterial actin MreB promotes local membrane fluidization

The bacterial actin homologue MreB plays a key role in rod cell shape determination. We recently showed that MreB from the Gram-positive bacterium Geobacillus stearothermophilus (MreBGs) polymerizes into straight pairs of protofilaments in the presence of both ATP and a lipid surface. Membrane interaction is thought to be mediated by electrostatic interactions with anionic lipids, with final anchoring relying on two spatially close hydrophobic motifs that protrude from the MreBGs monomers, forming a putative membrane-insertion domain. Here, we determined the binding properties of ATP and ADP to MreBGs using fluorescence anisotropy, and monitored ATP-mediated binding and polymer formation on lipid bilayers using liposome binding assays and AFM, respectively. Finally, we used solid-state NMR to visualize the interaction between the membrane and MreBGs at the atomic level. Our findings reveal that MreBGs has similar affinity for both ATP and ADP, unlike eukaryotic actin. We also show that monomeric MreBGs establishes peripheral contacts with the membrane likely through electrostatic interactions, while ATP-induced MreBGs filaments insert into the lipid bilayer without interfering with the membrane lamellar phase and have a significant local fluidifying effect. Statement of significanceBacteria rely on the actin-like protein MreB to determine and maintain their cell shape, like actin does in eukaryotic cells. To perform its tightly regulated morphogenetic function, MreB forms membrane-associated nanofilaments in vivo, which control the cell wall biosynthetic machinery. We recently demonstrated that, in vitro, MreB from the Gram-positive bacterium Geobacillus stearothermophilus requires both ATP and lipids to polymerize into pairs of filaments. Here, we show that in the presence of ATP, Geobacillus MreB forms membrane-bound filaments that directly impact local membrane fluidity, which could translate into a regulatory effect on cell wall synthetic enzymes. We further reveal that MreB binds both ATP and ADP with similar affinity, unlike eukaryotic actin, which preferentially binds ATP over ADP, and speculate that this could be a mechanism modulating the pool of polymerization-competent MreB in bacteria.

biophysics↗

bioRxiv2026

Optimizing actin turnover in cell-like conditions

A dynamic actin cytoskeleton, characterized by rapid filament turnover, is essential for driving intracellular transport and cellular movement. Despite extensive study, the contributions of many actin-binding proteins (ABPs) that regulate the different steps of the actin turnover cycle (filament assembly, disassembly and recycling into polymerizable actin monomers) remain ill-defined. Here, we introduce novel sensitive in vitro assays to quantitatively assess how ABPs catalyze actin turnover. By accurately measuring nucleotide exchange dynamics and ATP consumption, these assays enable robust characterization of ABP activity across broad concentration ranges. Using these methods and modeling of these reactions, we systematically examined the contributions of five conserved regulators, both individually and in combination, and identified conditions that maximize turnover efficiency. We also determined that increasing F-actin concentration to cellular levels affects ABP activity. Finally, we demonstrated that rapid actin turnover is preserved during encapsulation in cell-sized vesicles using the cDICE method. Together, these advances provide versatile tools and new insights into actin cytoskeletal dynamics under physiologically relevant conditions.

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

Evolutionarily diverged on-switch for actin assembly in fungal endocytosis

Clathrin-mediated endocytosis is a conserved eukaryotic trafficking process where an Arp2/3 complex nucleated branched actin network provides force for vesicle formation. The mechanisms that initiate endocytic actin assembly are incompletely understood. In the fission yeast, Schizosaccharomyces pombe, actin assembly is initiated by Dip1, an Arp2/3 activator. In the budding yeast, Saccharomyces cerevisiae, the initiation of actin assembly has remained a mystery. Here we show that S. cerevisiae Ldb17, the homolog of Dip1, functions as an on-switch for endocytic actin assembly. Unexpectedly, the regulation of Ldb17 is more complicated than that of constitutively active Dip1. Ldb17 is controlled by a coat protein, Sla1, via separate recruitment and activation steps. This regulation was likely lost in the S. pombe lineage and this simplification may be related to other changes in actin assembly between these species. Our findings add a key missing piece in the understanding of endocytosis in S. cerevisiae and reveal an intriguing evolutionary tinkering of the actin on-switch.

cell biology↗

bioRxiv2024

Cell polarity control by an unconventional G-protein complex in bacteria

In Myxococcus xanthus, cell polarity and motility are regulated by the small GTPase MglA and its associated regulators, MglB, a potential GTPase Activating Protein and the RomRX complex, a potential Guanine nucleotide Exchange Factor. However, recent results have questioned the exact function of these regulators. Using a new type of fluorescent nucleotides for fluorescence-based anisotropy, we first demonstrate that RomRX does not function as a GEF but as an effector that binds strictly to MglAGTP. Secondly, based on an enzymology model, we found that MglB functions as a weak catalyst, unlike typical GAPs, and requires additional factors to dissociate from MglA. We demonstrate that RomRX, and specifically RomR, effectively competes with MglA by sequestering MglB. This low affinity interaction is still permissive for GAP activity but sufficient to partition these proteins in vivo as indicated by a biomimetic assay in oil emulsion droplets reconstituting these interactions in a cell-sized environment. These results suggest a new model for cell polarity where RomRX exerts a dual function, targeting MglAGTP to the pole via its effector function and preventing its accumulation at the lagging pole by destabilizing the MglA-MglB complex.

biochemistry↗

bioRxiv2023

Unusual actin-binding mechanism and the role of profilin in actin dynamics of trypanosomatid parasites

Diseases caused by Leishmania, and Trypanosoma parasites, such as leishmaniasis and African sleeping sickness, are a major health problem in tropical countries. Due to their complex life cycle involving both vertebrate and insect hosts, and > 1 billion years of evolutionarily distance, the cell biology of these trypanosomatid parasites exhibits pronounced differences to animal cells. For example, the actin cytoskeleton of trypanosomatids is highly divergent when compared to the other eukaryotes. To understand how actin dynamics are regulated in trypanosomatid parasites, we focused on a central actin-binding protein profilin. Co-crystal structure of Leishmania major actin in complex with L. major profilin revealed that, although the overall folds of actin and profilin are conserved in eukaryotes, Leishmania profilin contains a unique -helical insertion, which interacts with the target binding cleft of actin monomer. This insertion is conserved across the Trypanosomatidae family, and is strikingly similar to the structure of WH2 domain, a small actin-binding motif found in many other cytoskeletal regulators. We demonstrate that the WH2-like motif contributes to actin monomer-binding and enhances the actin nucleotide exchange activity of Leishmania profilin. Surprisingly, unlike other profilins characterized so far, Leishmania profilin inhibited formin-catalyzed actin filament assembly in a mechanism that is dependent on the presence of the WH2-like motif. By generating profilin knockout and knockin Leishmania mexicana strains, we show that profilin is important for efficient endocytic sorting in parasites, and that the ability to bind actin monomers and proline-rich proteins, as well as the presence of a functional WH2-like motif, are important for the in vivo function of Leishmania profilin. Collectively, this study uncovers the molecular principles by which actin dynamics are regulated by profilin in trypanosomatids. Moreover, the unusual actin-binding mechanism of profilin identified here could be applied for designing inhibitors against pathogenic trypanosomatid parasites. AUTHOR SUMMARYLeishmania and Trypanosoma parasites are a major health problem as they cause various diseases in humans and other vertebrates. Currently, there are no specific drugs to treat the diseases caused by these trypanosomatid parasites. Similar to all other eukaryotes, trypanosomatid parasites have an actin cytoskeleton, which is essential for the viability of parasites. Interestingly, both actin and actin-regulatory machineries of these parasites are highly divergent from the ones of animals, making them possible drug targets to treat diseases caused by these parasites. To uncover how the actin cytoskeleton of trypanosomatid parasites is regulated, we focused on a central actin-binding protein, profilin. Importantly, our experiments revealed that trypanosomatid profilins interact with actin through a different structural mechanism as compared to animal profilins, and have specific effects on the assembly of actin filaments. Our genetic studies demonstrate that these specific features of trypanosomatid profilin are also critical for the proper function on this protein in parasites. Our study provides new insight into the cell biology of trypanosomatid parasites. We also envision that the structural and functional differences between trypanosomatid and human profilins can be applied for developing compounds for selectively neutralizing Leishmania and Trypanosoma parasites.

biochemistry↗

bioRxiv2021

Specialization of actin isoforms derived from the loss of key interactions with regulatory factors

A paradox of eukaryotic cells is that while some species assemble a complex actin cytoskeleton from a single ortholog, other species utilize a greater diversity of actin isoforms. The physiological consequences of using different actin isoforms, and the molecular mechanisms by which highly conserved actin isoforms are segregated into distinct networks, are poorly known. Here, we sought to understand how a simple biological system, composed of a unique actin and a limited set of actin-binding proteins, reacts to a switch to heterologous actin expression. Using yeast as a model system and biomimetic assays, we show that such perturbation causes drastic reorganization of the actin cytoskeleton. Our results indicate that defective interaction of a heterologous actin for important regulators of actin assembly limits certain actin assembly pathways while reinforcing others. Expression of two heterologous actin variants, each specialized in assembling a different network, rescues cytoskeletal organization and confers resistance to external perturbation. Hence, while species using a unique actin have homeostatic actin networks, actin assembly pathways in species using several actin isoforms may act more independently.

cell biology↗

bioRxiv2019

Mammalian Amoeboid Swimming is propelled by molecular and not protrusion-based paddling in Lymphocytes

Mammalian cells developed two main migration modes. The slow mesenchymatous mode, like fibroblasts crawling, relies on maturation of adhesion complexes and actin fiber traction, while the fast amoeboid mode, observed exclusively for leukocytes and cancer cells, is characterized by weak adhesion, highly dynamic cell shapes, and ubiquitous motility on 2D and in 3D solid matrix. In both cases, interactions with the substrate by adhesion or friction are widely accepted as a prerequisite for mammalian cell motility, which precludes swimming. We show here experimentally and computationally that leukocytes do swim, and that propulsion is not fueled by waves of cell deformation but by a rearward and inhomogeneous treadmilling of the cell envelope. We model the propulsion as a molecular paddling by transmembrane proteins linked to and advected by the actin cortex, whereas freely diffusing transmembrane proteins hinder swimming. This mechanism explains that swimming is five times slower than the cortex retrograde flow. Resultantly the ubiquitous ability of mammalian amoeboid cells to migrate in various environments can be explained for lymphocytes by a single machinery of envelope treadmilling.

biophysics↗

bioRxiv2018

The Range of Cooperativity Modulates Actin Binding Protein Cluster Size, Density and Dynamics

The actin cytoskeleton is composed of multiple networks which are specialized for several processes such as cell motility or cell division. Each of these networks are composed of organized actin microfilaments which are decorated with specific sets of actin binding proteins (ABPs). The molecular mechanisms guiding ABPs to specific actin networks are still poorly understood, but cooperativity, the mechanism by which the binding of an ABP is positively influenced by proximal bound ABPs, plays a crucial role in generating locally dense stretches of ABPs. Cooperative binding is characterized by its amplitude, but also by the range at which its effects are propagated along an actin filament through long-range allosteric interactions. The range of these allosteric effects is still debated, but is likely to be significant at the lengthscale of actin filaments in cells. Here, we investigated how cooperativity influences the clustering of ABPs, using a stochastic computational model of binding of ABPs to actin filaments. The model reproduces the formation of ABP clusters observed experimentally at the single filament scale, and provides a theoretical estimation of the range of cooperativity for proteins such as ADF/cofilin. We found that both the amplitude and the spatial range of cooperativity dramatically impact the properties of clustering. However, the parameters of cooperativity modulate differently the rate of assembly, size and dynamics of the ABP clusters, suggesting that cooperativity is an efficient mechanism to regulate precisely the recruitment of ABPs in cells. This work provides a more general framework for future understanding of how actin networks acquire distinct and specific protein compositions from a common cytoplasm.

biophysics↗

bioRxiv2018

Conditions for optimal mechanical power generation by a bundle of growing actin filaments

Bundles of actin filaments are central to a large variety of cellular structures, such as filopodia, stress fibers, cytokinetic rings or focal adhesions. The mechanical properties of these bundles are critical for proper force transmission and force bearing. Previous mathematical modeling efforts have focused on bundles rigidity and shape. However, it remains unknown how bundle length and thickness are controlled by external physical factors, and how the attachment of the bundle to a load affects its ability to transmit forces. In this paper, we present a biophysical model for dynamic bundles of actin filaments that takes into account individual filaments, their interaction with each other and with an external load. In combination with in vitro motility assays of beads coated with formins, our model allowed us to characterize conditions for bead movement and bundle buckling. From the deformation profiles, we determined key biophysical properties of tethered actin bundles, such as their rigidity and filament density. Our model also demonstrated that filaments undulate under lateral constraints applied by external forces or by neighboring filaments of the bundle. Last, our model allowed us to identify optimum conditions in filament density and barbed end tethering to the load for a maximal yield of mechanical power by a dynamic actin bundle.

biophysics↗