Biology subjects

Casuso, I.

Publications and source records attributed to Casuso, I..

6 recordsLinked to original sources

bioRxiv2026

Stepwise assembly and mechanical stability of nsP3 Helical Scaffolds in Chikungunya Virus

Chikungunya virus (CHIKV) is a positive-sense, single-stranded RNA virus that replicates its genome through a viral replication complex (RC) essential for successful infection. The RC consists of four non-structural proteins: nsP1, nsP2, nsP3, and nsP4. In this study, we focused on nsP3, which comprises three distinct domains: the alphavirus unique domain (AUD), the long and intrinsically disordered hypervariable domain (HVD), and the enzymatic macrodomain (MD). Notably, nsP3 assembles into helicoidal macrostructures, termed helical scaffolds (HSs), which are components of the CHIKV replication complex and are involved in the formation of macromolecular traps in the cytoplasm of the infected cell called Alphagranules (AGs). HSs form tubular helical scaffolds assembled through the AUD, with the MD and HVD flexibly deployed to the scaffold exterior. Despite their functional importance, the mechanisms governing HS formation remain poorly understood. Using High-Speed Atomic Force Microscopy (HS-AFM) and Electron Microscopy (EM) we achieved new findings on the structure, mechanics and assembly of these macrostructures. Our results show the nsP3 monomers follow intermediate steps before completing the assembly into full helical scaffolds, with each nsP3 domain contributing differently to this process. The macrodomain (MD) provides mechanical stability, yet it fractures under tens of piconewtons of force. This rigidity acts as a mechanical regulator that constrains the position of the HVD, which in turn mediates interactions with host factors and neighboring HSs. Through this interplay, nsP3 achieves both structural cohesion and functional adaptability, promoting the higher-order organization of HSs into honeycomb-like networks.

molecular biology↗

bioRxiv2026

Lysenin Induced Membrane Curvature on Erythrocytes and Nanoporous Mica Free Standing Membranes

Beta porin toxins are virulence factors that oligomerize in host cell membranes, forming beta-barrel pores that permeabilize target cells, leading to osmotic imbalance, cell lysis, and immune evasion. Despite extensive research on beta-pore-forming toxins (PFTs), significant gaps remain in understanding their role as actors of large-scale supramolecular membrane remodeling. While beta-PFTs are traditionally viewed as passive cylindrical conduits, their biological efficacy is fundamentally tied to an evolved ability to structurally alter the host cell membrane. In this study, we combine erythrocytes, model membranes and a novel engineered nanopores on mica substrates. We overcome the limitation of traditional mica substrates and demonstrate that lysenin pores do not merely permeabilize the membrane; they curve it into rigid, dome-like supramolecular structures. Moreover, to gain biophysical insight beyond the standard membrane models which do not directly consider the membrane insertions, we propose a hybrid model combining the biophysical models and the architectural models of pendentive domes, where the intrinsic wedge-shaped geometry of the monomers is considered. Experimental mechanical assessment of the lysenins domes shows a non-linear mechanical of stiffness with respect the radius of curvature as predicted by our hybrid model. Collectively, our work suggests that the beta-PFTs induce membrane curvature by the assembly of wedges. Beyond their pore-forming function, this membrane-bending activity may represent an additional mechanism by which beta-PFTs interfere with host-cell membrane repair.

biophysics↗

bioRxiv2025

A Membrane-Disruptive Action of VBIT-4 Challenges Its Role as a Widely Used VDAC Oligomerization Inhibitor

Voltage-dependent anion channel (VDAC) is the most abundant protein of the mitochondrial outer membrane and a key regulator of metabolite exchange and mitochondrial physiology. Its oligomerization has been proposed to control processes such as mitochondrial DNA release and membrane remodeling, yet the underlying mechanisms remain poorly defined. VBIT-4 has been widely used over the last decade as a putative inhibitor of VDAC1 oligomerization, despite limited mechanistic validation. Here, using high-speed atomic force microscopy (AFM), we directly visualized VDAC1 assemblies in lipid membranes and examined the effect of VBIT-4. Unexpectedly, VBIT-4 induced membrane defects and permeabilization at micromolar concentrations, independently of VDAC1. Quantitative AFM analysis further shows that VBIT-4 does not alter VDAC1 cluster organization. Complementary electrophysiology, microscale thermophoresis, and coarse-grained molecular dynamics demonstrate that VBIT-4 partitions into lipid bilayers, increases membrane permeability, and destabilizes membrane structure, without detectable effects on VDAC1 channel properties or assemblies. Consistent with this mechanism, VBIT-4 induces VDAC1-independent cytotoxicity in HeLa cells at concentrations above 10 {micro}M. Together, these results demonstrate that VBIT-4 does not act as a specific inhibitor of VDAC1 oligomerization but instead functions as a membrane-active compound. This work provides a revised framework for interpreting studies using VBIT-4 and highlights the importance of systematically assessing drug-membrane interactions when targeting membrane proteins.

biochemistry↗

bioRxiv2024

Lipid composition of the membrane governs the oligomeric organization of VDAC1

VDACs, the most abundant proteins in the outer mitochondrial membrane (MOM), are crucial for mitochondrial physiology. VDAC regulate metabolite and ion exchange, modulate calcium homeostasis, and play roles in numerous cellular events such as apoptosis, mitochondrial DNA (mtDNA) release, and different diseases. Mitochondrial function is closely tied to VDAC oligomerization, influencing key processes like mtDNA release and apoptosis, but the molecular drivers of this oligomerization remain unclear. In this study, we investigate the effects of three major MOM lipids on VDAC assemblies using atomic force microscopy and molecular dynamics simulations. Our results show that phosphatidylethanolamine and cholesterol regulate VDAC assembly, with the formation of stable lipid-protein organization of various size and compaction. Deviations from physiological lipid content disrupted native-like VDAC assemblies, highlighting the importance of lipid environment in VDAC organization. These findings underscore how lipid heterogeneity and changes in membranes influence VDAC function.

biochemistry↗

bioRxiv2021

Atomic reconstruction of biomolecular structures from AFM images and quantitative validation of experimental data using simulated AFM scanning

We provide the BioAFMviewer-Toolbox, an extension of our previously developed software platform for simulated AFM scanning of biomolecular structures and dynamics. The focus was on developing a toolbox of methods which employ simulated AFM scanning combined with quantitative analysis to facilitate the interpretation of resolution-limited AFM images. The key advancement is the automatized fitting of biomolecular structures to experimental AFM images, which allows to reconstruct 3D atomistic structures from AFM surface scans. Moreover, several methods for detailed analysis and comparison of surface topographies in simulated and experimental AFM images are provided. We demonstrate the applicability of the developed tools in the interpretation of high-speed AFM observations of proteins. The toolbox is implemented into the versatile interactive interface of the BioAFMviewer, which is a free software package available at www.bioafmviewer.com.

biophysics↗

bioRxiv2021

Structure and dynamics of FCHo2 docking on membranes

Clathrin-mediated endocytosis (CME) is a central trafficking pathway in eukaryotic cells regulated by phosphoinositides. The plasma membrane phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) plays an instrumental role in driving CME initiation. The F-BAR domain only protein 1 and 2 complex (FCHo1/2) is among the early proteins that reach the plasma membrane, but the exact mechanisms triggering its recruitment remains elusive. Here, we show the molecular dynamics of FCHo2 self-assembly on membranes by combining bottom-up synthetic approaches on in vitro and cellular membranes. Our results indicate that PI(4,5)P2 domains assist FCHo2 docking at specific membrane regions, where it self-assembles into ring-like shape protein patches. We show that binding of FCHo2 on cellular membranes promotes PI(4,5)P2 clustering at the boundary of cargo receptors and that this accumulation enhances clathrin assembly. Thus, our results provide a mechanistic framework that could explain the recruitment of early PI(4,5)P2-interacting proteins at endocytic sites.

biophysics↗