Biology subjects

Bally, M.

Publications and source records attributed to Bally, M..

12 recordsLinked to original sources

bioRxiv2026

SIZE DETERMINATION AND MULTIPLEXED FLUORESCENCE-BASED PHENOTYPING OF SINGLE CELL-DERIVED MEMBRANE VESICLES USING A NANOFLUIDIC DEVICE

Extracellular vesicles (EVs) are cell-secreted biological nanoparticles that play a crucial role in intercellular communication and are gaining increasing attention as diagnostic biomarkers, therapeutic agents, and drug delivery vehicles. Consequently, the development of robust and sensitive methods for their characterization is essential. Herein we present the use of a microscope-mounted nanofluidic device for direct size determination and multi-parametric (3-color) fluorescence-based phenotyping of single biological nanoparticles that are in the size range of 20-200 nm in a method we denote Nano-SMF (SMF; size and multiplexed fluorescence). We demonstrate that it is possible to accurately determine the size of nanoparticles by analyzing their one-dimensional Brownian motion during directional flow through nanochannels, achieving size distributions for monodisperse nanoparticle solutions that are on par with TEM analysis, and size discrimination of nanoparticle mixtures that is significantly improved compared to conventional nanoparticle tracking analysis (NTA). Furter, we demonstrate that the method can be applied to analyze EVs directly in minute volumes of cell supernatant, avoiding pre-isolation or concentration steps. The method was applied to phenotype CD63- and CD81-positive EVs from a human embryonic kidney cell model, demonstrating that vesicle sub-populations defined by these two tetraspanin biomarkers differ significantly in size.

biophysics↗

bioRxiv2025

Mechanisms involved in the lipid membrane tubulation activity of the pH-responsive MakA protein from Vibrio cholerae

Bacterial pore-forming toxins (PFTs) are key virulence factors that disrupt host cell membranes. The Vibrio cholerae cytotoxin MakA, a member of the ClyA structural family of -PFTs, exhibits a unique, pH-dependent ability to induce lipid membrane tubulation. This study elucidates the molecular mechanisms underlying this process. We identify the N-terminal histidine residue (His30) of MakA as a critical pH sensor that is indispensable for membrane tubulation but not required for membrane binding and cell lysis. Substitution of His30 with lysine, MakAH30K , abrogated tubulation without affecting membrane insertion, instead triggering aberrant membrane fusion events. Circular dichroism spectroscopy analysis revealed that the MakAH30K substitution attenuates the robust -helical transition observed in wild-type MakA upon lipid membrane binding, providing a molecular explanation for its functional defect. Furthermore, we established that membrane cholesterol contributes to MakA-induced tubulation of lipid membranes, as its absence or mutation of a putative cholesterol-binding motif MakAI236D&I237D abolishes both membrane association and tubule formation. A key implication of our work is the mechanistic uncoupling of membrane tubulation from cytolysis, revealing that MakA can perturb membranes via distinct pathways. These findings define a sophisticated activation mechanism where His30 protonation in the acidic host environment and specific cholesterol binding synergistically drive the conformational change necessary for helical oligomerization and membrane tubulation. We discuss our findings from the viewpoint of possible convergent evolution, where bacterial PFTs like MakA partly may mimic ancient membrane remodelers involved in membrane repair pathways, such as ESCRT-III, but for bacterial fitness in host interactions and pathogenesis. Moreover, the present study provides a foundation for possible development of therapeutic strategies aimed at inhibiting these critical interactions.

biochemistry↗

bioRxiv2025

METTL3 regulates exocytosis independently of m6A

RNA modification pathways are often mis-regulated in various cancers, with N6-methyladenosine (m6A) having a pivotal role in cancer progression and metastasis. Methyltransferase-like 3 (METTL3), a core component of the m6A methyltransferase complex, functions not only as an m6A writer but also promotes tumorigenesis through m6A-independent mechanisms. Here, we show that METTL3 is mislocalized to the cytoplasm in breast cancer tumors from patients, contributing to the oncogenic phenotype. Cytoplasmic METTL3 interacts with EXOC7, a key regulator of exocytosis, promoting its stabilization. Additionally, METTL3 regulates m6A-dependent alternative splicing of EXOC7. Silencing METTL3 impairs vesicle trafficking and the breast cancer secretome - effects that do not rely on its enzymatic activity but instead involve METTL3-mediated stabilization of EXOC7 and potentially other exocyst components. Furthermore, METTL3 knockdown impairs invadopodia formation, collagen matrix invasion, and focal adhesion morphology in vitro, while inhibition of METTL3 catalytic activity does not. Our findings uncover non-catalytic roles of METTL3 in regulating exocytosis and the cancer secretome.

molecular biology↗

bioRxiv2025

Lipid packing frustration in the outer leaflet of the plasma membrane prevents scission of caveolae

Lipid packing is a fundamental characteristic of bilayer membranes. It affects all membrane-associated processes ranging from curvature generation to membrane fission. Yet, we lack detailed mechanistic understanding of how lipid packing directly affects these processes in cellular membranes. Here, we address this by focusing on caveolae, small 0-shaped invaginations of the plasma membrane which serve as key regulators of cellular lipid sorting and mechano-responses. In addition to caveolae coat proteins, the lipid membrane is a core component of caveolae that critically impacts both the biogenesis, morphology and stability of such membrane invaginations. We show that the small compound Dyngo-4a adsorbs and inserts into the membrane, resulting in a dramatic dynamin-independent inhibition of caveola dynamics. Analysis of model membranes in combination with molecular dynamics simulations revealed that a substantial amount of Dyngo-4a was inserted and positioned at the level of cholesterol in the bilayer affecting lipid order in a cholesterol dependent manner. Dyngo-4a-treatment resulted in decreased lipid packing of the plasma membrane. This prevented caveolae internalization and lateral diffusion without affecting their morphology, associated proteins, or the overall cell stiffness. Artificially increasing plasma membrane cholesterol levels was found to counteract the block in caveola dynamics caused by Dyngo-4a-mediated lipid packing frustration. Therefore, we propose that the outer leaflet lipid packing of cholesterol in the the plasma membrane critically contributes to the confinement of caveolae to the plasma membrane. Significance statementLarsson et al., demonstrate that lipid packing critically impacts the stability of small 0-shaped plasma membrane cavities termed caveolae. The plasma membrane (PM) release of caveolae is dynamin independent and halted by PM incorporation of the small compound Dyngo-4a, which leads to decreased lipid packing as characterized by the authors. The block in caveola internalization can be counteracted by increased PM levels of cholesterol which is enriched in caveolae and increases the lipid packing. This proof-of-principle study shows that lipid packing controls membrane budding generating membrane vesicles that remain stably associated with the membrane for an extended period of time.

cell biology↗

bioRxiv2024

Site-specific sulfations regulate the physicochemical properties of papillomavirus-heparan sulfate interactions for entry

Certain human papillomaviruses (HPVs) are etiological agents for several anogenital and oropharyngeal cancers. During initial infection, HPV16, the most prevalent cancer-causing type, specifically interacts with heparan sulfates (HS), not only enabling initial cell attachment but also triggering a crucial conformational change in viral capsids termed structural activation. It is unknown, whether such HS-HPV16 interactions depend on HS sulfation patterns. Thus, we probed potential roles of HS sulfations using cell-based functional and physicochemical assays, including single molecule force spectroscopy. Our results demonstrate that N-sulfation of HS is crucial for virus binding and structural activation by providing high affinity sites, and that additional 6O-sulfation is required to mechanically stabilize the interaction, whereas 2O-sulfation and 3O-sulfation are mostly dispensable. Together, our findings identify the contribution of HS sulfation patterns to HPV16 binding and structural activation and reveal how distinct sulfation groups of HS synergize to facilitate HPV16 entry, which, in turn, likely influences the tropism of HPVs. TeaserDistinct heparan sulfations facilitate HPV16 binding and structural activation through their physico-chemical properties.

microbiology↗

bioRxiv2024

Variant-specific interactions at the plasma membrane: Heparan sulfate's impact on SARS-CoV-2 binding kinetics

The worldwide spread of SARS-CoV-2 has been characterised by the emergence of several variants of concern (VOCs) presenting an increasing number of mutations in the viral genome. The spike glycoprotein, responsible for engaging the viral receptor ACE2, exhibits the highest density of mutations, suggesting an ongoing evolution to optimize viral entry. However, previous studies focussed on isolated molecular interactions, neglecting the intricate composition of the plasma membrane and the interplay between viral attachment factors. Our study explores the role of avidity and of the complexity of the plasma membrane composition in modulating the virus-host binding kinetics during the early stages of viral entry for the original Wuhan strain and three VOCs: Omicron BA.1, Delta, and Alpha. We employ fluorescent liposomes decorated with spike from several VOCs as virion mimics in single-particle tracking studies on native supported lipid bilayers derived from pulmonary Calu-3 cells. Our findings reveal an increase in the affinity of the multivalent bond to the cell surface for Omicron driven by an increased association rate. We show that heparan sulfate (HS), a sulfated glycosaminoglycan commonly expressed on cells plasma membrane, plays a central role in modulating the interaction with the cell surface and we observe a shift in its role from screening the interaction with ACE2 in early VOCs to an important binding factor for Omicron. This is caused by a [~]10-fold increase in Omicrons affinity to HS compared to the original Wuhan strain, as shown using atomic force microscopy-based single-molecule force spectroscopy. Our results show the importance of coreceptors, particularly HS, and membrane complexity in the modulation of the attachment in SARS-CoV-2 VOCs. We highlight a transition in the variants attachment strategy towards the use of HS as an initial docking site, which likely plays a role in shaping Omicrons tropism towards infection of the upper airways, milder symptoms, and higher transmissibility.

microbiology↗

bioRxiv2023

Efficient clathrin-mediated entry of enteric adenoviruses in human duodenal cells

Enteric adenovirus types F40 and 41 (EAdVs) are a leading cause of diarrhea and diarrhea-associated death in young children and have recently been proposed to cause acute hepatitis in children. Unlike other adenoviruses, EAdVs exhibit hitherto a strict tropism for gastrointestinal tissues with, to date, unknown infection mechanism and target cells. In this study, we turn to potentially limiting host factors by comparison of EAdV entry in cell lines with respiratory and intestinal origin by cellular perturbation, virus particle tracking and transmission electron microscopy. Our analyses highlight kinetic advantages in duodenal HuTu80 cell infection and reveal a larger fraction of mobile particles, faster virus uptake and infectious particle entry in intestinal cells. Moreover, EAdVs display a dependence on clathrin- and dynamin-dependent pathways in intestinal cells. Detailed knowledge of virus entry routes and host factor requirements is essential to understand pathogenesis and develop new countermeasures. Hence, this study provides novel insights into the entry mechanisms of a medically important virus with emerging tropism in a physiologically relevant cell line. Author SummaryEnteric adenoviruses have historically been difficult to grow in cell culture, which resulted in lack of knowledge of host factors and pathways required for infection of these medically relevant viruses. Previous studies in non-intestinal cell lines showed slow infection kinetics and generated comparatively low virus yields compared to other adenovirus types. We suggest duodenum derived HuTu80 cells as a superior cell line for studies to complement efforts using complex intestinal tissue models. We show that viral host cell factors required for virus entry differ between cell lines from distinct origins and demonstrate the importance of clathrin-mediated endocytosis.

microbiology↗

bioRxiv2023

Recruitment of apolipoprotein E facilitates Herpes simplex virus 1 release

Over two decades, epidemiological studies have revealed that interactions between human polymorphic apolipoprotein 4 (ApoE, isoform 4) and herpes simplex virus type 1 (HSV1) associate with higher risk of Alzheimers disease, a serious and increasing issue among elder populations worldwide. Nevertheless, little is known about the mechanisms behind ApoE-HSV1 interactions at molecular levels. Here, we investigate the effects of ApoE on the HSV1 infectious life cycle in in vitro cell experiments. Analysis of HSV1 growth curves shows that HSV1 production is promoted in presence of any of the three ApoE isoforms, with ApoE 3 or 4 demonstrating more proviral effects than ApoE 2. Quantification by qPCR reveals that the presence of ApoE 2, 3, or 4 leads to an increase of HSV1 extracellular release but unchanged levels of viral genome copies within cells or on the cell surface, indicating that virus replication, assembly, or transport to cell membrane are not affected. Further test of virus release directly demonstrates that HSV1 detachment from the cell surface is promoted by ApoE. Subsequent results reveal that ApoE is both present in purified HSV1 particles produced in ApoE-expressing cells after ultra-centrifugation and able to incorporate into HSV1 particles after purification, suggesting that harbouring ApoE may play a key role in the pro-viral effect of ApoE. Along these lines, we tested the infectious behaviour of ApoE-coated viruses and observed faster attachment kinetics and higher entry efficiencies of ApoE decorated HSV1. Our hypothesis that the association with ApoE leads to modified interactions of the virus with the cell membrane during entry and egress, was further validated in biophysical experiments. In such experiments, HSV1-membrane interaction kinetics and apparent affinity between HSV1 and native supported lipid bilayers (a plasma membrane mimic) were quantified using total internal reflection microscopy. HSV1 particles decorated with ApoE demonstrate both higher association (kon) and dissociation rate constants (koff), as well as less irreversible binding to the membrane, which is in line with the biological experiments. Overall, our results provide new insights into the roles of ApoE during HSV1 infections, which is worth to be considered when studying their involvement during Alzheimers disease development.

microbiology↗

bioRxiv2022

Simultaneous Membrane and RNA Binding by Tick-Borne Encephalitis Virus Capsid Protein

Tick-borne encephalitis virus is an enveloped, pathogenic, RNA virus in the family Flaviviridae, genus Flavivirus. Viral particles are formed when the nucleocapsid, consisting of an RNA genome and multiple copies of the capsid protein, buds through the endoplasmic reticulum membrane and acquires the viral envelope and the associated proteins. The coordination of the nucleocapsid components to the sites of assembly and budding are poorly understood. Here, we investigate nucleocapsid assembly by characterizing the interactions of the wild-type and truncated capsid proteins with membranes by using biophysical methods and model membrane systems. We show that capsid protein initially binds membranes via electrostatic interactions with negatively-charged lipids which is followed by membrane insertion. Additionally, we show that membrane-bound capsid protein can recruit viral genomic RNA. We confirm the biological relevance of the biophysical findings by using mass spectrometry to show that purified virions contain negatively-charged lipids. Our results suggest that nucleocapsid assembly is coordinated by negatively-charged membrane patches on the endoplasmic reticulum and that the capsid protein mediates direct contacts between the nucleocapsid and the membrane.

biochemistry↗

bioRxiv2021

Protein-lipid interaction at low pH induces oligomerisation of the MakA cytotoxin from Vibrio cholerae

Many pathogenic bacteria produce protein toxins that target and perturb host cell membranes. The secreted -pore-forming toxins (-PFTs) cause membrane damage via pore formation. This study demonstrates a remarkable, hitherto unknown mechanism by an -PFT protein from Vibrio cholerae. As part of the MakA/B/E tripartite toxin, MakA is involved in membrane pore formation similar to other -PFTs. In contrast, MakA protein alone induces tube-like structures in the acidic lysosomal host cell compartment. In vitro studies unravel the dynamics of tubular growth, which occur in a pH-, lipid- and concentration-dependent manner. A 3.7-[A] cryo-electron microscopy structure of MakA filaments reveals a unique protein-lipid superstructure. In its active -PFT conformation, MakA embeds its transmembrane helices into a thin annular lipid bilayer and spirals around a central cavity. Our study provides molecular insights into a novel tubulation mechanism of an -PFT protein, revealing a new mode of action by a secreted bacterial toxin.

cell biology↗

bioRxiv2021

A tripartite cytolytic toxin formed by Vibrio cholerae proteins with flagellum-facilitated secretion

The protein MakA was discovered as a motility-associated secreted toxin from Vibrio cholerae, Here, we show that MakA is part of a gene cluster encoding four additional proteins: MakB, MakC, MakD and MakE. The MakA, MakB and MakE proteins were readily detected in culture supernatants of wild type V. cholerae whereas secretion was very much reduced from a flagellum deficient mutant. Crystal structures of MakA, MakB and MakE revealed structural relationship to a superfamily of bacterial pore-forming proteins. Cloning and expression of MakA/B/E in Escherichia coli resulted in toxicity of the bacteria towards Caenorhabditis elegans used as a predatory organism model. None of these Mak proteins alone or in pairwise combinations were cytolytic but an equimolar mixture of MakA, MakB and MakE acted as a tripartite cytolytic toxin in vitro causing lysis of erythrocytes and cytotoxicity on cultured human colon carcinoma cells. Formation of oligomeric complexes on liposomes was observed by electron microscopy. Oligomer interaction with membranes was initiated by MakA membrane binding followed by MakB and MakE joining in formation of a pore structure. A predicted membrane insertion domain of MakA was shown by site-directed mutagenesis to be essential for toxicity towards C. elegans. Bioinformatic analyses revealed that the makCDBAE gene cluster is present as a novel genomic island in the vast majority of sequenced genomes of V. cholerae and the fish pathogen V. anguillarum. We suggest that the hitherto unrecognized cytolytic MakA/B/E toxin can contribute to Vibrionaceae fitness and virulence potential in different host environments and organisms. Significance StatementVibrio cholerae, responsible for outbreaks and pandemics of cholera disease, is a highly motile organism by virtue of a single flagellum. We describe that the flagellum facilitates the secretion of three V. cholerae proteins encoded by a hitherto unrecognized novel genomic island. The proteins MakA/B/E can form a tripartite cytolytic toxin that lyses erythrocytes and is cytotoxic to cultured human cells. A structural basis for the Mak protein cytolytic activity was obtained by X-ray crystallography. Flagellum-facilitated secretion, remarkably ensuring spatially co-ordinated delivery of Mak proteins, revealed a new role for the V. cholerae flagellum considered of particular significance for the bacterial environmental persistence. Our findings will pave the way for the development of new diagnostics and therapeutic strategies against pathogenic Vibrionaceae.

biochemistry↗

bioRxiv2019

The ESCRT-III Isoforms CHMP2A And CHMP2B Display Different Effects On Membranes Upon Polymerization

ESCRT-III proteins are involved in many membrane remodeling processes including multivesicular body biogenesis as first discovered in yeast. In humans, CHMP2 exists as two potential isoforms, CHMP2A and CHMP2B, but their physical characteristics have not been compared yet. Here, we use a combination of technics on biomimetic systems and purified proteins to study their affinity and effects on membranes. We establish that CHMP2B binding is enhanced in the presence of PI(4,5)P2 lipids. In contrast, CHMP2A does not display lipid specificity and requires CHMP3 for binding significantly to membranes. On the micrometer scale and at moderate bulk concentrations, CHMP2B forms a reticular structure on membranes whereas CHMP2A (+CHMP3) binds homogeneously. Eventually, CHMP2A and CHMP2B unexpectedly induce different mechanical effects to membranes: CHMP2B strongly rigidifies them while CHMP2A (+CHMP3) has no significant effect. Altogether, we conclude that CHMP2B and CHMP2A cannot be considered as isoforms and might thus contribute differently to membrane remodeling processes.

biophysics↗