Biology subjects

Bonn, M.

Publications and source records attributed to Bonn, M..

7 recordsLinked to original sources

bioRxiv2026

The structured hairpin region of the bacterial ESCRT-III protein IM30 orchestrates stress-induced condensate formation

Biomolecular condensates are well characterized in eukaryotes, but their role in bacteria remains largely elusive. In the cyanobacteriumn Synechocystis sp. PCC 6803, the protein IM30, a member of the ESCRT-III superfamily of membrane remodeling proteins, forms stress-induced puncta across diverse environmental challenges, indicating a general adaptive response. Live-cell imaging reveals that IM30 is uniformly distributed throughout the cytoplasm at low concentrations but assembles into puncta upon exceeding a critical saturation threshold, a hallmark of liquid-liquid phase separation. Crucially, stress triggers puncta formation even below this threshold, suggesting stress lowers the phase-separation barrier. Super-resolution microscopy confirms spherical, condensate-like morphologies, while FRAP demonstrates rapid fluorescence recovery, consistent with with a fluid interior and dynamic exchange between puncta and the cytosol. Cellular IM30 levels exceed the in vitro determined critical concentration, placing the protein in a supersaturated state primed for condensation. Domain mapping identifies the structured 1-3 helical hairpin as the minimal phase separation driver; in contrast, the disordered 4-6 segment alone cannot phase-separate. Phase separation occurs within a physiologically relevant pH range (4.5-6.5), matching the localized acidification of the cyanobacterial cytoplasm at damaged thylakoid membranes. This directly links membrane stress, pH changes, and IM30 recruitment. Collectively, these findings establish IM30 puncta as bona fide, stress-responsive biomolecular condensates that function as rapid stress sensors and effectors, providing a mechanistic framework for phase separation and condensate formation by bacterial ESCRT-III proteins during environmental adaptation.

biochemistry↗

bioRxiv2025

Membrane binding of a cyanobacterial ESCRT-III protein crucially involves the helix α1-3 hairpin conserved in all superfamily members

IM30, the inner membrane-associated protein of 30 kDa (also known as Vipp1) is essential for thylakoid membrane biogenesis and/or maintenance in chloroplasts and cyanobacteria. IM30 and its bacterial homolog PspA belong to the ESCRT-III superfamily, proteins previously thought to be restricted to eukaryotes and archaea. Despite low sequence similarity, IM30 shares key structural and functional features with eukaryotic ESCRT-IIIs, including a conserved 1-2 helical hairpin core and the ability to form oligomeric barrel- or rod assemblies that mediate membrane remodeling. Using IM30 variants, we now show that initial membrane recruitment of IM30 is driven by electrostatic interactions between the positively charged 1-3 helical hairpin and negatively charged lipid surfaces, paralleling the role of charged helical regions in some eukaryotic ESCRT-IIIs. This likely initiates lateral assembly of IM30 into higher-order barrel or rod structures on the membrane. Once assembled, 0 helices within these oligomers engage and stabilize internalized membrane tubules, mirroring membrane interaction strategies of eukaryotic ESCRT-IIIs, which use both N-terminal sequences and charged residues on 1/2. Thus, our findings demonstrate a conserved membrane binding and remodeling mechanism across the ESCRT-III superfamily, underscoring an evolutionary link in membrane dynamics between pro- and eukaryotes. SignificanceIM30, a membrane-associated protein found in cyanobacteria and chloroplasts, along with its bacterial homolog PspA, belongs to the ESCRT-III superfamily. Despite low sequence conservation, these proteins share structural and functional features with eukaryotic ESCRT-III proteins. We show that IM30 binds membranes via a conserved structural motif, followed by lateral assembly into higher-order complexes. This supports a mechanism of membrane remodeling that is conserved in prokaryotic and eukaryotic members of the ESCRT-III superfamily.

biochemistry↗

bioRxiv2023

Structural adaptability and surface activity oftardigrade-inspired peptides

Tardigrades are unique micro-animals that withstand harsh conditions, such as extreme temperatures and desiccation. Recently, it was found that specific cytoprotective proteins are essential for ensuring this high environmental tolerance. In particular, cytoplasmic abundant heat soluble (CAHS) proteins, which are intrinsically disordered, adopt more ordered conformations upon desiccation, and are involved in the vitrification of the cytoplasm. The design and synthesis short peptides capable of mimicking the structural behavior (and thus the cytoprotective properties) of CAHS proteins would be beneficial for potential biomedical applications, including the development of novel heat-resistant preservatives for sensitive drug formulations. As a first step in this direction, we selected several model peptides of varying lengths derived from the conserved CAHS motifs 1 and 2, which are part of the intrinsically disordered CAHS-c2 region. We then studied their structures using circular dichroism and linear and two-dimensional infrared spectroscopy in the presence of the desolvating agent TFE (2,2,2-trifluoroethanol), which mimics desiccation. We found that the CAHS model peptides are mostly disordered at 0% TFE (a result that we confirmed by molecular dynamics simulations), but adopt a more -helical structure upon the addition of the desolvating agent, similar to what is observed for full CAHS proteins. Additionally, we employed sum frequency generation to investigate the surface activity of the peptides at the air/water interface to mimic a partial dehydration effect. Interestingly, all model peptides are surface active and also adopt a helical structure at the air/water interface. Thus, the selected sequences represent promising model peptides that show similarities in the physicochemical behavior to full CAHS proteins. Our results also suggest that arginine might be a crucial element in defining the strong propensity of these peptides to adopt a helical structure. In the future, the use CAHS model peptides to design new synthetic peptide-based materials could make it possible to mimic and exploit the cytoprotective properties of naturally occurring tardigrade proteins. SIGNIFICANCETardigrades are micro-animals that can survive extreme conditions such as desiccation and high temperatures. Recent work has shown that this capability is related to the presence of specific proteins that can remodel in order to protect the organisms cells. Mimicking this behavior using small peptides that preserve the structural properties of the full proteins is highly desirable in potential biomedical applications, such as the storage of heat-sensitive drugs. Here, we study the structural properties of model peptides derived from the conserved region of cytoplastic tardigrade proteins, and show that these peptides preserve some of the conformational behavior of the full protein under drying conditions. These peptides can therefore be used as a starting point for the design of synthetic model systems based on tardigrade-inspired peptides for tailored applications.

biochemistry↗

bioRxiv2023

A solid beta-sheet structure is formed at the surface of FUS liquid droplets during aging

Insights into liquid droplet formation via liquid-liquid phase separation and the subsequent liquid-to-solid phase transition are important for understanding cell dynamics, as well as a number of neurodegenerative disorders. We report here, using the example of the FUsed in Sarcoma (FUS) protein, an investigation of the liquid droplet maturation process combining solution- and solid-state NMR spectroscopy, Raman spectroscopy, and light and electron microscopies. Our study reveals that the surface of the droplets plays a critical role in this process. Indeed, when comparing a biphasic sample, in which liquid droplets are stabilized in an agarose matrix, with a pure monophasic condensed phase sample, we find that the liquid-droplet maturation kinetics is faster in the biphasic FUS sample, owing to the larger surface-to-volume ratio. In addition, using Raman spectroscopy, we observe structural differences upon liquid-droplet maturation between the inside and the surface of liquid droplets, which is of {beta}-sheet content as revealed by solid-state NMR. This is detected very early on and increases over time. In agreement with these observations, a solid crust-like shell is visually seen by microaspiration experiments. After several months, electron microscopy reveals that the matured FUS droplets have converted into solid linear fibrils distinct from the fibril core of seeded fibrils reported previously, as arginine side-chains from the arginine-and-glycine-rich domain (RGG) motif are partially rigidified, highlighting the participation of this motif in the liquid-to-solid transition. In presence of RNA, this aging process is not taking place.

biochemistry↗

bioRxiv2020

Effect of shear and tensile loading on fibrin molecular structure revealed by coherent Raman microscopy

Blood clots are essential biomaterials that prevent blood loss and provide a temporary scaffold for tissue repair. In their function, these materials must be capable of resisting mechanical forces from hemodynamic shear and contractile tension without rupture. Fibrin networks, the primary load-bearing element in blood clots, have unique nonlinear mechanical properties resulting from their hierarchical structure, which provides multiscale load bearing from fiber deformation to protein unfolding. Here, we study the fiber and molecular scale response of fibrin under shear and tensile loads in situ using a combination of fluorescence and vibrational (molecular) microscopy. Imaging protein fiber orientation and molecular vibrations, we find that fiber orientation and molecular changes in fibrin appear at much larger strains under shear compared to uniaxial tension. Orientation levels reached at 150% shear strain were reached already at 60% tensile strain, and molecular unfolding of fibrin was only seen at shear strains above 300%, whereas fibrin unfolding began already at 20% tensile strain. Moreover, shear deformation caused progressive changes in vibrational modes consistent with increased protofibril and fiber packing that were already present even at very low tensile deformation. Together with a bioinformatic analysis of the fibrinogen primary structure, we propose a scheme for the molecular response of fibrin from low to high deformation, which may relate to the teleological origin of its resistance to shear and tensile forces. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/205005v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@d72dfborg.highwire.dtl.DTLVardef@10bed75org.highwire.dtl.DTLVardef@12d33aorg.highwire.dtl.DTLVardef@1e9b40f_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Tension causes structural unfolding of intracellular intermediate filaments

Intermediate filament (IF) proteins are a class of proteins that constitute different filamentous structures in mammalian cells. As such, IF proteins are part of the load-bearing cytoskeleton and support the nuclear envelope. Molecular dynamics simulations have shown that IF proteins undergo secondary structural changes to compensate mechanical loads, which has been confirmed by experimental in vitro studies on IF hydrogels. However, the structural response of intracellular IF to mechanical load has yet to be elucidated in cellulo. Here, we use in situ nonlinear Raman imaging combined with multivariate data analysis to quantify the intracellular secondary structure of the IF cytoskeletal protein vimentin under different states of cellular tension. We find that cells under native cellular tension contain more unfolded vimentin than chemically or physically relaxed specimens. This indicates that unfolding of IF proteins occurs intracellularly when sufficient forces are applied, suggesting that IF structures act as local force sensors in the cell to mark locations under large mechanical tension.

biophysics↗

bioRxiv2018

Quantitative mapping of triacylglycerol chain length and saturation using broadband CARS microscopy

Lipid droplets (LDs) are highly dynamic organelles that store neutral lipids, primarily triacylglycerols (TAGs), and are found in many cell types. While their primary function is to store excess energy, LDs are also modified in different disease states and during developmental processes. In many cases, not only the presence, but also the composition, of LDs can be equally important. In humans, LD composition has been linked to diseases such as type 2 diabetes; in plants and yeast, LD composition is relevant for engineering these organisms into biological factories in, e.g., algal bioenergy or food oil production. Therefore, lipid analysis of biological LDs with high speed and accuracy in situ is a very active area of research. Here we present an approach for in situ, quantitative TAG analysis using label-free, coherent Raman microscopy to decipher LD TAG composition in different biochemically complex samples. Our method allows direct visualization of inter-LD compositional heterogeneity of physiological quantities - TAG chain length and number of C=C bonds - with sub-micrometer spatial resolution within 5-100 milliseconds. Combined with virtually no sample preparation, this approach should enable rapid and accurate TAG LD analysis for a variety of applications.

bioengineering↗