Biology subjects

Bano, F.

Publications and source records attributed to Bano, F..

6 recordsLinked to original sources

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

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↗

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↗

bioRxiv2020

Membrane binding controls ordered self-assembly of animal septins

Septins are conserved cytoskeletal proteins that regulate cell cortex mechanics. The mechanisms of their interactions with the plasma membrane remain poorly understood. Here we show by cell-free reconstitution that membrane binding requires electrostatic interactions of septins with anionic lipids and promotes the ordered self-assembly of fly septins into filamentous meshworks. Transmission electron microscopy reveals that both fly and mammalian septins form arrays of single and paired filaments. Atomic force microscopy and quartz crystal microbalance demonstrate that the fly filaments form mechanically rigid, 12 to 18 nm thick, double layers of septins. By contrast, C-terminally truncated septin mutants form 4 nm thin monolayers, indicating that stacking requires the C-terminal coiled coils on DSep2 and Pnut subunits. Our work shows that membrane binding is required for fly septins to form ordered arrays of single and paired filaments and provides new insights into the mechanisms by which septins may regulate cell surface mechanics.

cell biology↗