Biology subjects

Aumont-Nicaise, M.

Publications and source records attributed to Aumont-Nicaise, M..

6 recordsLinked to original sources

bioRxiv2026

Interlaboratory benchmarking of differential scanning fluorimetry reveals sources of variability in protein thermal stability parameters

Differential scanning fluorimetry (DSF) is widely used to assess protein thermal stability and to compare protein variants, ligands, or formulations with limited sample consumption. Yet, the reproducibility of DSF derived parameters across laboratories, instruments and analysis workflows remains poorly defined. Here, we report a two phase interlaboratory benchmark using a shared VHH sample across 26 laboratories, 28 instruments and seven DSF instrument models. In Phase 1, participants applied their routine acquisition and analysis workflows, whereas in Phase 2, acquisition conditions were harmonised. Datasets were analysed locally and then centrally reprocessed with MoltenProt. Participant reported apparent melting temperatures (Tm) showed good within instrument repeatability but moderate inter instrument dispersion. Harmonisation of acquisition conditions did not substantially reduce this dispersion. Conversely, centralised reprocessing narrowed the distribution among retained datasets, indicating contributions from analysis workflows and data acceptance criteria in addition to instrument dependent factors. Comparison with differential scanning calorimetry (DSC) further showed that apparent Tm depended strongly on the fluorescence readout. For this VHH, single wavelength fluorescence at 350 nm showed the closest agreement with the DSC transition temperature, whereas the 350/330 nm ratio and barycentric mean generally yielded higher values. Furthermore, DSF derived apparent unfolding enthalpies were more variable than apparent Tm. Together, these findings show that DSF can be highly repeatable within a defined instrument and workflow, but that quantitative comparability across instruments depends on instrument specific effects, the choice of fluorescence readout and analysis strategy. Improved comparability will require transparent reporting of acquisition and analysis conditions, explicit data quality criteria, and well characterised reference materials to monitor instrument dependent offsets.

biophysics↗

bioRxiv2026

Ecological Diversification into Broad Thermal Niches Revealed by Protein Resurrection and Proteome-Wide Ancestral Reconstruction During the Rapid Radiation of Alvinellid Worms

In the deep Pacific and Indian oceans, Alvinellid worms diversified about 100 million years ago to colonize a variety of hydrothermal vent environments. It has been suggested that the last common ancestor of this family was a thermophilic species. However, the evolutionary history of these worms is complex, with putative gene flow among ancestors. In this study, we investigated the early evolution of the family in relation to the diversification of thermal niches. We demonstrated that phylogenetic histories of alvinellid species greatly vary along chromosomes, possibly due to allele introgression between nascent ecotypes. We performed sequence reconstructions of ancestral cytosolic MDH and Cu/Zn SOD under the two most frequent phylogenies encountered along the genome. In silico simulations of folding stability for these two enzymes were highly correlated with their biophysical characterization (micro-calorimetry and differential scanning fluorimetry), and were used to predict the thermostability of thousands of alternatively reconstructed proteins. We further generalized the reconstruction at the proteome scale, taking the amino-acid usage bias as a proxy for folding stability in a new phylogenetic model accounting for amino-acid variations over time. Both approaches agree that the last common ancestors of Alvinellidae gained highly stable proteins, comparable to proteins of present-day thermotolerant species. Our computational protocol allowed us to validate this scenario under multiple phylogenetic hypotheses and different sequence reconstruction models. Considered together, Alvinellidae shed light on how metazoan species diversify in order to colonize different thermal niches, combining adaptive mutation and selection on genetic variants. SignificanceDeep-sea hydrothermal vents are among the most extreme environments on Earth. They, however, represent oases of life for a restricted number of highly specialized species. As such, Alvinellid worms constitute an exceptional family, including highly thermotolerant animals such as the Pompeii worm. Despite their vent endemicity, Alvinellidae thrive under contrasting ecological conditions, particularly regarding temperature. Understanding how this family diversified to colonize broad thermal regimes is of major importance for dissecting the mechanisms by which species adapt in highly unstable environments. Combining experimental protein resurrection, proteome-wide predictions, and new phylogenetic models, we assess the reliability of ancestral sequence reconstructions that represent molecular thermometers to evaluate past environmental conditions.

evolutionary biology↗

bioRxiv2023

The Bacterial Replicative Helicase Loader DciA is a DNA Condenser.

The loading of the bacterial replicative helicase is an essential step for genome replication and depends on the assistance of accessory proteins. Several of these proteins have been identified across the bacterial phyla. DciA is the most common loading protein in bacteria, yet the one whose mechanism is the least understood. We have previously shown that VcDciA from Vibrio cholerae, composed of a globular KH-like domain followed by an unfolded extension, has a strong affinity for DNA. Here, we characterized the droplets formed by VcDciA upon interaction with a short single-stranded substrate. We demonstrate the fluidity of these droplets using light microscopy and address their network organization through electron microscopy, thereby bridging events to conclude on a liquid-liquid phase separation behavior. Additionally, we observe the recruitment of VcDnaB inside the VcDciA-DNA droplets. We show that DnaC from Escherichia coli is also competent to form these condensate structures in the presence of ssDNA. Our data open up possibilities for the involvement of DciA in the formation of non-membrane compartments within the bacterium, facilitating the assembly of replication players with the chromosomal DNA.

biochemistry↗

bioRxiv2022

DNA methylation at a single cytosine embedded in the W-box cis-element repels binding of WRKY transcription factors through steric hindrance

Previously, we showed that the Arabidopsis active demethylase ROS1 prunes DNA methylation at the promoters of a subset of immune-responsive genes to facilitate their transcriptional activation during antibacterial defence (Halter et al., 2021). In particular, ROS1 was shown to demethylate the RLP43 promoter in a region carrying a functional W-box cis-element, thereby ensuring a tight binding of WRKY transcriptional factors (TFs) onto DNA. Here, we first extend these findings by showing that DNA methylation at W-box elements decreases the binding of several Arabidopsis WRKY TFs in vitro. Furthermore, we provide evidence that DNA methylation at a single cytosine located in the W-box of the RLP43 promoter strongly repels DNA binding of an Arabidopsis WRKY TF in vitro. Using structural modelling, we demonstrate that this cytosine interacts through van der Waals contacts with the conserved tyrosine of WRKY DNA binding domains. Significantly, our model predicts steric hindrance when a 5-methyl group is present on this specific cytosine, thereby likely preventing tight binding of WRKY DNA binding domains. Finally, because the WRKY motif and the residues involved in DNA contacts are conserved, we propose that this methylation-dependent WRKY-DNA binding inhibitory mechanism must be widespread across plant species.

plant biology↗

bioRxiv2021

Structural convergence for tubulin binding of CPAP and vinca domain microtubule inhibitors

Microtubule dynamics is regulated by various cellular proteins and perturbed by small molecule compounds. To what extent the mechanism of the former resembles that of the latter is an open question. We report here structures of tubulin bound to the PN2-3 domain of CPAP, a protein controlling the length of the centrioles. We show that an -helix of the PN2-3 N-terminal region binds and caps the longitudinal surface of the tubulin {beta} subunit. Moreover, a PN2-3 N-terminal stretch lies in a {beta}-tubulin site also targeted by fungal and bacterial peptide-like inhibitors of the vinca domain, sharing a very similar binding mode with these compounds. Therefore, our results identify several characteristic features of cellular partners that bind to this site and highlight a structural convergence of CPAP with small molecule inhibitors of microtubule assembly.

biochemistry↗

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↗