Publications by authors named "David Alsteens"

91 Publications

Probing PIEZO1 Localization upon Activation Using High-Resolution Atomic Force and Confocal Microscopy.

Nano Lett 2021 06 14;21(12):4950-4958. Epub 2021 Jun 14.

Louvain Institute of Biomolecular Science and Technology, Université Catholique de Louvain, Louvain-la-Neuve 1348, Belgium.

PIEZO1 ion channels are activated by mechanical stimuli, triggering intracellular chemical signals. Recent structural studies suggest that plasma membrane tension or local curvature changes modulate PIEZO1 channel gating and activation. However, whether PIEZO1 localization is governed by tension gradients or long-range mechanical perturbations across the cells is still unclear. Here, we probe the nanoscale localization of PIEZO1 on red blood cells (RBCs) at high resolution (∼30 nm), and we report for the first time the existence of submicrometric PIEZO1 clusters in native conditions. Upon interaction with Yoda1, an allosteric modulator, PIEZO1 clusters increase in abundance in regions of higher membrane tension and lower curvature. We further show that PIEZO1 ion channels interact with the spectrin cytoskeleton in both resting and activated states. Our results point toward a strong interplay between plasma membrane tension gradients, curvature, and cytoskeleton association of PIEZO1.
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http://dx.doi.org/10.1021/acs.nanolett.1c00599DOI Listing
June 2021

Author Correction: Submolecular probing of the complement C5a receptor-ligand binding reveals a cooperative two-site binding mechanism.

Commun Biol 2021 May 12;4(1):592. Epub 2021 May 12.

Université catholique de Louvain, Louvain Institute of Biomolecular Science and Technology, 1348, Louvain-la-Neuve, Belgium.

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http://dx.doi.org/10.1038/s42003-021-02174-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8116332PMC
May 2021

Liquid-Liquid Phase Separation Enhances TDP-43 LCD Aggregation but Delays Seeded Aggregation.

Biomolecules 2021 04 8;11(4). Epub 2021 Apr 8.

Department of Neurosciences, Experimental Neurology and Leuven Brain Institute (LBI), KU Leuven-University of Leuven, 3000 Leuven, Belgium.

Aggregates of TAR DNA-binding protein (TDP-43) are a hallmark of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Although TDP-43 aggregates are an undisputed pathological species at the end stage of these diseases, the molecular changes underlying the initiation of aggregation are not fully understood. The aim of this study was to investigate how phase separation affects self-aggregation and aggregation seeded by pre-formed aggregates of either the low-complexity domain (LCD) or its short aggregation-promoting regions (APRs). By systematically varying the physicochemical conditions, we observed that liquid-liquid phase separation (LLPS) promotes spontaneous aggregation. However, we noticed less efficient seeded aggregation in phase separating conditions. By analyzing a broad range of conditions using the Hofmeister series of buffers, we confirmed that stabilizing hydrophobic interactions prevail over destabilizing electrostatic forces. RNA affected the cooperativity between LLPS and aggregation in a "reentrant" fashion, having the strongest positive effect at intermediate concentrations. Altogether, we conclude that conditions which favor LLPS enhance the subsequent aggregation of the TDP-43 LCD with complex dependence, but also negatively affect seeding kinetics.
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http://dx.doi.org/10.3390/biom11040548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8068378PMC
April 2021

Editorial: Scanning Probe Microscopies and Related Methods in Biology.

Front Mol Biosci 2021 29;8:657939. Epub 2021 Mar 29.

Department of Biosystems Science and Engineering, ETH Zurich, Zurich, Switzerland.

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http://dx.doi.org/10.3389/fmolb.2021.657939DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8044836PMC
March 2021

Reovirus directly engages integrin to recruit clathrin for entry into host cells.

Nat Commun 2021 04 12;12(1):2149. Epub 2021 Apr 12.

Louvain Institute of Biomolecular Science and Technology, Université catholique de Louvain, Louvain-la-Neuve, Belgium.

Reovirus infection requires the concerted action of viral and host factors to promote cell entry. After interaction of reovirus attachment protein σ1 with cell-surface carbohydrates and proteinaceous receptors, additional host factors mediate virus internalization. In particular, β1 integrin is required for endocytosis of reovirus virions following junctional adhesion molecule A (JAM-A) binding. While integrin-binding motifs in the surface-exposed region of reovirus capsid protein λ2 are thought to mediate integrin interaction, evidence for direct β1 integrin-reovirus interactions and knowledge of how integrins function to mediate reovirus entry is lacking. Here, we use single-virus force spectroscopy and confocal microscopy to discover a direct interaction between reovirus and β1 integrins. Comparison of interactions between reovirus disassembly intermediates as well as mutants and β1 integrin show that λ2 is the integrin ligand. Finally, using fluidic force microscopy, we demonstrate a functional role for β1 integrin interaction in promoting clathrin recruitment to cell-bound reovirus. Our study demonstrates a direct interaction between reovirus and β1 integrins and offers insights into the mechanism of reovirus cell entry. These results provide new perspectives for the development of efficacious antiviral therapeutics and the engineering of improved viral gene delivery and oncolytic vectors.
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http://dx.doi.org/10.1038/s41467-021-22380-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8041799PMC
April 2021

Impaired Cytoskeletal and Membrane Biophysical Properties of Acanthocytes in Hypobetalipoproteinemia - A Case Study.

Front Physiol 2021 23;12:638027. Epub 2021 Feb 23.

CELL Unit & PICT Imaging Platform, de Duve Institute, UCLouvain, Brussels, Belgium.

Familial hypobetalipoproteinemia is a metabolic disorder mainly caused by mutations in the gene. In its homozygous form it can lead without treatment to severe ophthalmological and neurological manifestations. In contrast, the heterozygous form is generally asymptomatic but associated with a low risk of cardiovascular disease. Acanthocytes or thorny red blood cells (RBCs) are described for both forms of the disease. However, those morphological changes are poorly characterized and their potential consequences for RBC functionality are not understood. Thus, in the present study, we asked whether, to what extent and how acanthocytes from a patient with heterozygous familial hypobetalipoproteinemia could exhibit altered RBC functionality. Acanthocytes represented 50% of the total RBC population and contained mitoTracker-positive surface patches, indicating the presence of mitochondrial fragments. While RBC osmotic fragility, calcium content and ATP homeostasis were preserved, a slight decrease of RBC deformability combined with an increase of intracellular free reactive oxygen species were observed. The spectrin cytoskeleton was altered, showing a lower density and an enrichment in patches. At the membrane level, no obvious modification of the RBC membrane fatty acids nor of the cholesterol content were detected but the ceramide species were all increased. Membrane stiffness and curvature were also increased whereas transversal asymmetry was preserved. In contrast, lateral asymmetry was highly impaired showing: (i) increased abundance and decreased functionality of sphingomyelin-enriched domains; (ii) cholesterol enrichment in spicules; and (iii) ceramide enrichment in patches. We propose that oxidative stress induces cytoskeletal alterations, leading to increased membrane stiffness and curvature and impaired lipid lateral distribution in domains and spicules. In addition, ceramide- and spectrin-enriched patches could result from a RBC maturation defect. Altogether, the data indicate that acanthocytes are associated with cytoskeletal and membrane lipid lateral asymmetry alterations, while deformability is only mildly impaired. In addition, familial hypobetalipoproteinemia might also affect RBC precursors leading to disturbed RBC maturation. This study paves the way for the potential use of membrane biophysics and lipid vital imaging as new methods for diagnosis of RBC disorders.
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http://dx.doi.org/10.3389/fphys.2021.638027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7940373PMC
February 2021

Single-Virus Force Spectroscopy Discriminates the Intrinsic Role of Two Viral Glycoproteins upon Cell Surface Attachment.

Nano Lett 2021 01 29;21(1):847-853. Epub 2020 Dec 29.

Université Catholique de Louvain, Louvain Institute of Biomolecular Science and Technology, Louvain-la-Neuve 1348, Belgium.

Viruses are one of the most efficient pathogenic entities on earth, resulting from millions of years of evolution. Each virus particle carries the minimum number of genes and proteins to ensure their reproduction within host cells, hijacking some host replication machinery. However, the role of some viral proteins is not yet unraveled, with some appearing even redundant. For example, murid herpesvirus 4, the current model for human gammaherpesvirus infection, can bind to cell surface glycosaminoglycans using both glycoproteins gp70 and gH/gL. Here, using atomic force microscopy, we discriminate their relative contribution during virus binding to cell surface glycosaminoglycans. Single-virus force spectroscopy experiments demonstrate that gH/gL is the main actor in glycosaminoglycan binding, engaging more numerous and more stable interactions. We also demonstrated that Fab antibody fragments targeting gH/gL or gp70 appear to be a promising treatment to prevent the attachment of virions to cell surfaces.
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http://dx.doi.org/10.1021/acs.nanolett.0c04609DOI Listing
January 2021

Stress-Induced Catch-Bonds to Enhance Bacterial Adhesion.

Trends Microbiol 2021 04 19;29(4):286-288. Epub 2020 Dec 19.

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Croix du Sud, 4-5, bte L7.07.07, B-1348 Louvain-la-Neuve, Belgium. Electronic address:

Physical forces have a profound influence on bacterial cell physiology and disease. A striking example is the formation of catch-bonds that reinforce under mechanical stress. While mannose-binding by the Escherichia coli FimH adhesin has long been the only thoroughly studied microbial catch-bond, it has recently become clear that proteins from other species, such as staphylococci, are also engaged in such stress-dependent interactions.
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http://dx.doi.org/10.1016/j.tim.2020.11.009DOI Listing
April 2021

Submolecular probing of the complement C5a receptor-ligand binding reveals a cooperative two-site binding mechanism.

Commun Biol 2020 12 18;3(1):786. Epub 2020 Dec 18.

Université catholique de Louvain, Louvain Institute of Biomolecular Science and Technology, 1348, Louvain-la-Neuve, Belgium.

A current challenge to produce effective therapeutics is to accurately determine the location of the ligand-biding site and to characterize its properties. So far, the mechanisms underlying the functional activation of cell surface receptors by ligands with a complex binding mechanism remain poorly understood due to a lack of suitable nanoscopic methods to study them in their native environment. Here, we elucidated the ligand-binding mechanism of the human G protein-coupled C5a receptor (C5aR). We discovered for the first time a cooperativity between the two orthosteric binding sites. We found that the N-terminus C5aR serves as a kinetic trap, while the transmembrane domain acts as the functional site and both contributes to the overall high-affinity interaction. In particular, Asp282 plays a key role in ligand binding thermodynamics, as revealed by atomic force microscopy and steered molecular dynamics simulation. Our findings provide a new structural basis for the functional and mechanistic understanding of the GPCR family that binds large macromolecular ligands.
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http://dx.doi.org/10.1038/s42003-020-01518-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749166PMC
December 2020

Label-Free Imaging of Cholesterol Assemblies Reveals Hidden Nanomechanics of Breast Cancer Cells.

Adv Sci (Weinh) 2020 Nov 8;7(22):2002643. Epub 2020 Oct 8.

Louvain Institute of Biomolecular Science and Technology (LIBST) Université catholique de Louvain Louvain-la-Neuve 1348 Belgium.

Tumor cells present profound alterations in their composition, structural organization, and functional properties. A landmark of cancer cells is an overall altered mechanical phenotype, which so far are linked to changes in their cytoskeletal regulation and organization. Evidence exists that the plasma membrane (PM) of cancer cells also shows drastic changes in its composition and organization. However, biomechanical characterization of PM remains limited mainly due to the difficulties encountered to investigate it in a quantitative and label-free manner. Here, the biomechanical properties of PM of a series of MCF10 cell lines, used as a model of breast cancer progression, are investigated. Notably, a strong correlation between the cell PM elasticity and oncogenesis is observed. The altered membrane composition under cancer progression, as emphasized by the PM-associated cholesterol levels, leads to a stiffening of the PM that is uncoupled from the elastic cytoskeletal properties. Conversely, cholesterol depletion of metastatic cells leads to a softening of their PM, restoring biomechanical properties similar to benign cells. As novel therapies based on targeting membrane lipids in cancer cells represent a promising approach in the field of anticancer drug development, this method contributes to deciphering the functional link between PM lipid content and disease.
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http://dx.doi.org/10.1002/advs.202002643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7675049PMC
November 2020

Atomic Force Microscopy-Based Force Spectroscopy and Multiparametric Imaging of Biomolecular and Cellular Systems.

Chem Rev 2020 Nov 9. Epub 2020 Nov 9.

Louvain Institute of Biomolecular Science and Technology, Université Catholique de Louvain (UCLouvain), Croix du Sud, 4-5, bte L7.07.07, B-1348 Louvain-la-Neuve, Belgium.

During the last three decades, a series of key technological improvements turned atomic force microscopy (AFM) into a nanoscopic laboratory to directly observe and chemically characterize molecular and cell biological systems under physiological conditions. Here, we review key technological improvements that have established AFM as an analytical tool to observe and quantify native biological systems from the micro- to the nanoscale. Native biological systems include living tissues, cells, and cellular components such as single or complexed proteins, nucleic acids, lipids, or sugars. We showcase the procedures to customize nanoscopic chemical laboratories by functionalizing AFM tips and outline the advantages and limitations in applying different AFM modes to chemically image, sense, and manipulate biosystems at (sub)nanometer spatial and millisecond temporal resolution. We further discuss theoretical approaches to extract the kinetic and thermodynamic parameters of specific biomolecular interactions detected by AFM for single bonds and extend the discussion to multiple bonds. Finally, we highlight the potential of combining AFM with optical microscopy and spectroscopy to address the full complexity of biological systems and to tackle fundamental challenges in life sciences.
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http://dx.doi.org/10.1021/acs.chemrev.0c00617DOI Listing
November 2020

Force-clamp spectroscopy identifies a catch bond mechanism in a Gram-positive pathogen.

Nat Commun 2020 10 27;11(1):5431. Epub 2020 Oct 27.

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Croix du Sud, 4-5, bte L7.07.07, B-1348, Louvain-la-Neuve, Belgium.

Physical forces have profound effects on cellular behavior, physiology, and disease. Perhaps the most intruiguing and fascinating example is the formation of catch-bonds that strengthen cellular adhesion under shear stresses. Today mannose-binding by the Escherichia coli FimH adhesin remains one of the rare microbial catch-bond thoroughly characterized at the molecular level. Here we provide a quantitative demonstration of a catch-bond in living Gram-positive pathogens using force-clamp spectroscopy. We show that the dock, lock, and latch interaction between staphylococcal surface protein SpsD and fibrinogen is strong, and exhibits an unusual catch-slip transition. The bond lifetime first grows with force, but ultimately decreases to behave as a slip bond beyond a critical force (~1 nN) that is orders of magnitude higher than for previously investigated complexes. This catch-bond, never reported for a staphylococcal adhesin, provides the pathogen with a mechanism to tightly control its adhesive function during colonization and infection.
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http://dx.doi.org/10.1038/s41467-020-19216-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7591895PMC
October 2020

Reovirus σ1 Conformational Flexibility Modulates the Efficiency of Host Cell Attachment.

J Virol 2020 11 9;94(23). Epub 2020 Nov 9.

Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee, USA

Reovirus attachment protein σ1 is a trimeric molecule containing tail, body, and head domains. During infection, σ1 engages sialylated glycans and junctional adhesion molecule-A (JAM-A), triggering uptake into the endocytic compartment, where virions are proteolytically converted to infectious subvirion particles (ISVPs). Further disassembly allows σ1 release and escape of transcriptionally active reovirus cores into the cytosol. Electron microscopy has revealed a distinct conformational change in σ1 from a compact form on virions to an extended form on ISVPs. To determine the importance of σ1 conformational mobility, we used reverse genetics to introduce cysteine mutations that can cross-link σ1 by establishing disulfide bonds between structurally adjacent sites in the tail, body, and head domains. We detected phenotypic differences among the engineered viruses. A mutant with a cysteine pair in the head domain replicates with enhanced kinetics, forms large plaques, and displays increased avidity for JAM-A relative to the parental virus, mimicking properties of ISVPs. However, unlike ISVPs, particles containing cysteine mutations that cross-link the head domain uncoat and transcribe viral positive-sense RNA with kinetics similar to the parental virus and are sensitive to ammonium chloride, which blocks virion-to-ISVP conversion. Together, these data suggest that σ1 conformational flexibility modulates the efficiency of reovirus host cell attachment. Nonenveloped virus entry is an incompletely understood process. For reovirus, the functional significance of conformational rearrangements in the attachment protein, σ1, that occur during entry and particle uncoating are unknown. We engineered and characterized reoviruses containing cysteine mutations that cross-link σ1 monomers in nonreducing conditions. We found that the introduction of a cysteine pair in the receptor-binding domain of σ1 yielded a virus that replicates with faster kinetics than the parental virus and forms larger plaques. Using functional assays, we found that cross-linking the σ1 receptor-binding domain modulates reovirus attachment but not uncoating or transcription. These data suggest that σ1 conformational rearrangements mediate the efficiency of reovirus host cell binding.
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http://dx.doi.org/10.1128/JVI.01163-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7654274PMC
November 2020

Molecular interaction and inhibition of SARS-CoV-2 binding to the ACE2 receptor.

Nat Commun 2020 09 11;11(1):4541. Epub 2020 Sep 11.

Louvain Institute of Biomolecular Science and Technology, Université Catholique de Louvain, Louvain-la-Neuve, Belgium.

Study of the interactions established between the viral glycoproteins and their host receptors is of critical importance for a better understanding of virus entry into cells. The novel coronavirus SARS-CoV-2 entry into host cells is mediated by its spike glycoprotein (S-glycoprotein), and the angiotensin-converting enzyme 2 (ACE2) has been identified as a cellular receptor. Here, we use atomic force microscopy to investigate the mechanisms by which the S-glycoprotein binds to the ACE2 receptor. We demonstrate, both on model surfaces and on living cells, that the receptor binding domain (RBD) serves as the binding interface within the S-glycoprotein with the ACE2 receptor and extract the kinetic and thermodynamic properties of this binding pocket. Altogether, these results provide a picture of the established interaction on living cells. Finally, we test several binding inhibitor peptides targeting the virus early attachment stages, offering new perspectives in the treatment of the SARS-CoV-2 infection.
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http://dx.doi.org/10.1038/s41467-020-18319-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486399PMC
September 2020

The Trypanosoma Brucei KIFC1 Kinesin Ensures the Fast Antibody Clearance Required for Parasite Infectivity.

iScience 2020 Aug 20;23(9):101476. Epub 2020 Aug 20.

Laboratory of Molecular Parasitology, IBMM, Université Libre de Bruxelles, 12, rue des professeurs Jeener et Brachet, 6041 Gosselies, Belgium. Electronic address:

Human innate immunity to Trypanosoma brucei involves the trypanosome C-terminal kinesin TbKIFC1, which transports internalized trypanolytic factor apolipoprotein L1 (APOL1) within the parasite. We show that TbKIFC1 preferentially associates with cholesterol-containing membranes and is indispensable for mammalian infectivity. Knockdown of TbKIFC1 did not affect trypanosome growth in vitro but rendered the parasites unable to infect mice unless antibody synthesis was compromised. Surface clearance of Variant Surface Glycoprotein (VSG)-antibody complexes was far slower in these cells, which were more susceptible to capture by macrophages. This phenotype was not due to defects in VSG expression or trafficking but to decreased VSG mobility in a less fluid, stiffer surface membrane. This change can be attributed to increased cholesterol level in the surface membrane in TbKIFC1 knockdown cells. Clearance of surface-bound antibodies by T. brucei is therefore essential for infectivity and depends on high membrane fluidity maintained by the cholesterol-trafficking activity of TbKIFC1.
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http://dx.doi.org/10.1016/j.isci.2020.101476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7479354PMC
August 2020

Multiparametric Atomic Force Microscopy Identifies Multiple Structural and Physical Heterogeneities on the Surface of .

ACS Omega 2020 Aug 13;5(33):20953-20959. Epub 2020 Aug 13.

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Croix du Sud, 4-5, bte L7.07.07, B-1348 Louvain-la-Neuve, Belgium.

A unique feature of the African trypanosome is the presence of an outer layer made of densely packed variable surface glycoproteins (VSGs), which enables the cells to survive in the bloodstream. Although the VSG coat is critical to pathogenesis, how exactly the glycoproteins are organized at the nanoscale is poorly understood. Here, we show that multiparametric atomic force microscopy is a powerful nanoimaging tool for the structural and mechanical characterization of trypanosomes, in a label-free manner and in buffer solution. Directly correlated images of the structure and elasticity of trypanosomes enable us to identify multiple nanoscale mechanical heterogeneities on the cell surface. On a ∼250 nm scale, regions of softer (Young's modulus ∼50 kPa) and stiffer (∼100 kPa) elasticity alternate, revealing variations of the VSG coat and underlying structures. Our nanoimaging experiments show that the cell surface is more heterogeneous than previously anticipated and offer promising prospects for the design of trypanocidal drugs targeting cell surface components.
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http://dx.doi.org/10.1021/acsomega.0c02416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7450619PMC
August 2020

Mechanochemical Activation of Class-B G-Protein-Coupled Receptor upon Peptide-Ligand Binding.

Nano Lett 2020 07 25;20(7):5575-5582. Epub 2020 Jun 25.

Louvain Institute of Biomolecular Science and Technology, Université Catholique de Louvain, Louvain-la-Neuve, Belgium.

Glucagon binding to the class-B G-protein-coupled glucagon receptor (GCGR) triggers the release of glucose from the liver during fasting. Recently, GCGR crystal structures have highlighted the conformation and molecular details of inactive and active receptor states. However, the dynamics of the conformational changes accompanying GCGR activation remains unclear. Here, we use multiplex force-distance curve-based atomic force microscopy (FD-based AFM) to probe glucagon binding to individual GCGRs and monitor dynamically the transition to the active conformer. After a "dock" step, in which glucagon is partially bound to the GCGR extracellular domain, further interactions of the N-terminus with the transmembrane domain trigger an increase in the stiffness of the complex, adopting a highly stable and rigid "lock" conformer. This mechanotransduction is key for G-protein recruitment.
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http://dx.doi.org/10.1021/acs.nanolett.0c02333DOI Listing
July 2020

Initial Step of Virus Entry: Virion Binding to Cell-Surface Glycans.

Annu Rev Virol 2020 09 12;7(1):143-165. Epub 2020 May 12.

Louvain Institute of Biomolecular Science and Technology, Université Catholique de Louvain, 1348 Louvain-la-Neuve, Belgium; email:

Virus infection is an intricate process that requires the concerted action of both viral and host cell components. Entry of viruses into cells is initiated by interactions between viral proteins and cell-surface receptors. Various cell-surface glycans function as initial, usually low-affinity attachment factors, providing a first anchor of the virus to the cell surface, and further facilitate high-affinity binding to virus-specific cell-surface receptors, while other glycans function as specific entry receptors themselves. It is now possible to rapidly identify specific glycan receptors using different techniques, define atomic-level structures of virus-glycan complexes, and study these interactions at the single-virion level. This review provides a detailed overview of the role of glycans in viral infection and highlights experimental approaches to study virus-glycan binding along with specific examples. In particular, we highlight the development of the atomic force microscope to investigate interactions with glycans at the single-virion level directly on living mammalian cells, which offers new perspectives to better understand virus-glycan interactions in physiologically relevant conditions.
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http://dx.doi.org/10.1146/annurev-virology-122019-070025DOI Listing
September 2020

Control of Ligand-Binding Specificity Using Photocleavable Linkers in AFM Force Spectroscopy.

Nano Lett 2020 05 27;20(5):4038-4042. Epub 2020 Apr 27.

Louvain Institute of Biomolecular Science and Technology, Université Catholique de Louvain, 1348 Louvain-la-Neuve, Belgium.

In recent decades, atomic force microscopy (AFM), in particular the force spectroscopy mode, has become a method of choice to study biomolecular interactions at the single-molecule level. However, grafting procedures as well as determining binding specificity remain challenging. We report here an innovative approach based on a photocleavable group that enables in situ release of the ligands bound to the AFM tip and thus allows direct assessment of the binding specificity. Applicable to a wide variety of molecules, the strategy presented here provides new opportunities to study specific interactions and deliver single molecules with high spatiotemporal resolution in a wide range of applications, including AFM-based cell biology.
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http://dx.doi.org/10.1021/acs.nanolett.0c01426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7252943PMC
May 2020

Endophilin-A3 and Galectin-8 control the clathrin-independent endocytosis of CD166.

Nat Commun 2020 03 19;11(1):1457. Epub 2020 Mar 19.

UCLouvain, Louvain Institute of Biomolecular Science and Technology, Group of Molecular Physiology, Croix du Sud 4-5, B-1348, Louvain-la-Neuve, Belgium.

While several clathrin-independent endocytic processes have been described so far, their biological relevance often remains elusive, especially in pathophysiological contexts such as cancer. In this study, we find that the tumor marker CD166/ALCAM (Activated Leukocyte Cell Adhesion Molecule) is a clathrin-independent cargo. We show that endophilin-A3-but neither A1 nor A2 isoforms-functionally associates with CD166-containing early endocytic carriers and physically interacts with the cargo. Our data further demonstrates that the three endophilin-A isoforms control the uptake of distinct subsets of cargoes. In addition, we provide strong evidence that the construction of endocytic sites from which CD166 is taken up in an endophilin-A3-dependent manner is driven by extracellular galectin-8. Taken together, our data reveal the existence of a previously uncharacterized clathrin-independent endocytic modality, that modulates the abundance of CD166 at the cell surface, and regulates adhesive and migratory properties of cancer cells.
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http://dx.doi.org/10.1038/s41467-020-15303-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7081352PMC
March 2020

Mechanical Forces between Mycobacterial Antigen 85 Complex and Fibronectin.

Cells 2020 03 14;9(3). Epub 2020 Mar 14.

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Croix du Sud, 4-5, bte L7.07.07, B-1348 Louvain-la-Neuve, Belgium.

Adhesion to extracellular matrix proteins is an important first step in host invasion, employed by many bacterial pathogens. In mycobacteria, the secreted Ag85 complex proteins, involved in the synthesis of the cell envelope, are known to bind to fibronectin (Fn) through molecular forces that are currently unknown. In this study, single-molecule force spectroscopy is used to study the strength, kinetics and thermodynamics of the Ag85-Fn interaction, focusing on the multidrug-resistant species. Single Ag85 proteins bind Fn with a strength of ~75 pN under moderate tensile loading, which compares well with the forces reported for other Fn-binding proteins. The binding specificity is demonstrated by using free Ag85 and Fn peptides with active binding sequences. The Ag85-Fn rupture force increases with mechanical stress (i.e., loading rate) according to the Friddle-Noy-de Yoreo theory. From this model, we extract thermodynamic parameters that are in good agreement with previous affinity determinations by surface plasmon resonance. Strong bonds (up to ~500 pN) are observed under high tensile loading, which may favor strong mycobacterial attachment in the lung where cells are exposed to high shear stress or during hematogenous spread which leads to a disseminated infection. Our results provide new insight into the pleiotropic functions of an important mycobacterial virulence factor that acts as a stress-sensitive adhesin.
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http://dx.doi.org/10.3390/cells9030716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140604PMC
March 2020

Glycan-mediated enhancement of reovirus receptor binding.

Nat Commun 2019 10 1;10(1):4460. Epub 2019 Oct 1.

Louvain Institute of Biomolecular Science and Technology, Université catholique de Louvain, Louvain-la-Neuve, Belgium.

Viral infection is an intricate process that requires the concerted action of both viral and host cell components. Entry of viruses into cells is initiated by interactions between viral proteins and their cell surface receptors. Despite recent progress, the molecular mechanisms underlying the multistep reovirus entry process are poorly understood. Using atomic force microscopy, we investigated how the reovirus σ1 attachment protein binds to both α-linked sialic acid (α-SA) and JAM-A cell-surface receptors. We discovered that initial σ1 binding to α-SA favors a strong multivalent anchorage to JAM-A. The enhanced JAM-A binding by virions following α-SA engagement is comparable to JAM-A binding by infectious subvirion particles (ISVPs) in the absence of α-SA. Since ISVPs have an extended σ1 conformer, this finding suggests that α-SA binding triggers a conformational change in σ1. These results provide new insights into the function of viral attachment proteins in the initiation of infection and open new avenues for the use of reoviruses as oncolytic agents.
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http://dx.doi.org/10.1038/s41467-019-12411-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6773860PMC
October 2019

Probing ligand-receptor bonds in physiologically relevant conditions using AFM.

Anal Bioanal Chem 2019 Oct 14;411(25):6549-6559. Epub 2019 Aug 14.

Louvain Institute of Biomolecular Science and Technology, Université catholique de Louvain, Croix du sud 4-5, 1348, Louvain-la-Neuve, Belgium.

Cell surface receptors, often called transmembrane receptors, are key cellular components as they control and mediate cell communication and signalling, converting extracellular signals into intracellular signals. Elucidating the molecular details of ligand binding (cytokine, growth factors, hormones, pathogens,...) to cell surface receptors and how this binding triggers conformational changes that initiate intracellular signalling is needed to improve our understanding of cellular processes and for rational drug design. Unfortunately, the molecular complexity and high hydrophobicity of membrane proteins significantly hamper their structural and functional characterization in conditions mimicking their native environment. With its piconewton force sensitivity and (sub)nanometer spatial resolution, together with the capability of operating in liquid environment and at physiological temperature, atomic force microscopy (AFM) has proven to be one of the most powerful tools to image and quantify receptor-ligand bonds in situ under physiologically relevant conditions. In this article, a brief overview of the rapid evolution of AFM towards quantitative biological mapping will be given, followed by selected examples highlighting the main advances that AFM-based ligand-receptor studies have brought to the fields of cell biology, immunology, microbiology, and virology, along with future prospects and challenges. Graphical abstract.
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http://dx.doi.org/10.1007/s00216-019-02077-6DOI Listing
October 2019

Regulatory Mechanisms of the Mucin-Like Region on Herpes Simplex Virus during Cellular Attachment.

ACS Chem Biol 2019 03 21;14(3):534-542. Epub 2019 Feb 21.

Department of Clinical Microbiology , Umeå University , 90185 Umeå , Sweden.

Mucin-like regions, characterized by a local high density of O-linked glycosylation, are found on the viral envelope glycoproteins of many viruses. Herpes simplex virus type 1 (HSV-1), for example, exhibits a mucin-like region on its glycoprotein gC, a viral protein involved in initial recruitment of the virus to the cell surface via interaction with sulfated glycosaminoglycans. So far, this mucin-like region has been proposed to play a key role in modulating the interactions with cellular glycosaminoglycans, and in particular to promote release of HSV-1 virions from infected cells. However, the molecular mechanisms and the role as a pathogenicity factor remains unclear. Using single virus particle tracking, we show that the mobility of chondroitin sulfate-bound HSV-1 virions is decreased in absence of the mucin-like region. This decrease in mobility correlates with an increase in HSV-1-chondroitin sulfate binding forces as observed using atomic force microscopy-based force spectroscopy. Our data suggest that the mucin-like region modulates virus-glycosaminoglycan interactions by regulating the affinity, type, and number of glycoproteins involved in the virus-glycosaminoglycan interaction. This study therefore presents new evidence for a role of the mucin-like region in balancing the interaction of HSV-1 with glycosaminoglycans and provides further insights into the molecular mechanisms used by the virus to ensure both successful cell entry and release from the infected cell.
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http://dx.doi.org/10.1021/acschembio.9b00064DOI Listing
March 2019

Seeing and sensing single G protein-coupled receptors by atomic force microscopy.

Curr Opin Cell Biol 2019 04 6;57:25-32. Epub 2018 Nov 6.

ETH Zürich, Department of Biosystems Science and Engineering, Mattenstrasse 26, 4058 Basel, Switzerland. Electronic address:

G protein-coupled receptors (GPCRs) relay extracellular information across cell membranes through a continuum of conformations that are not always captured in structures. Hence, complementary approaches are required to quantify the physical and chemical properties of the dynamic conformations linking to GPCR function. Atomic force microscopy (AFM)-based high-resolution imaging and force spectroscopy are unique methods to scrutinize GPCRs and to sense their interactions. Here, we exemplify recent AFM-based applications to directly observe the supramolecular assembly of GPCRs in native membranes, to measure the ligand-binding free-energy landscape, and how interactions modulate the structural properties of GPCRs. Common trends in GPCR function are beginning to emerge. We envision that technical developments in combining AFM with superresolution fluorescence imaging will provide insights into how cellular states modulate GPCRs and vice versa.
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http://dx.doi.org/10.1016/j.ceb.2018.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6472649PMC
April 2019

Multivalent binding of herpesvirus to living cells is tightly regulated during infection.

Sci Adv 2018 08 17;4(8):eaat1273. Epub 2018 Aug 17.

Louvain Institute of Biomolecular Science and Technology, Université catholique de Louvain, 1348 Louvain-la-Neuve, Belgium.

Viral infection, initiated by the landing of a virion on a cellular surface, is largely defined by the preliminary interactions established between viral particles and their receptors at the cell surface. While multiple parallel interactions would allow strong virus attachment, a low number of bonds could be preferred to allow lateral diffusion toward specific receptors and to promote efficient release of progeny virions from the cell surface. However, so far, the molecular mechanisms underlying the regulation of the multivalency in virus attachment to receptors are poorly understood. We introduce a new method to force-probe multivalent attachment directly on living cells, and we show, for the first time, direct evidence of a new mechanism by which a herpesvirus surface glycoprotein acts as a key negative regulator in the first step of herpesvirus binding. Using atomic force microscopy, we probe at the single-virion level the number and the strength of the bonds established with heparan sulfate both on model surfaces and on living cells. Our biophysical results, correlated with other techniques, show that the major envelope glycoprotein functions as a regulator of binding valency during both attachment and release steps, determining the binding, diffusion, and release potential of virions at the cellular surface.
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http://dx.doi.org/10.1126/sciadv.aat1273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6097811PMC
August 2018

A molecular mechanism for Wnt ligand-specific signaling.

Science 2018 08 19;361(6403). Epub 2018 Jul 19.

Laboratory of Neurovascular Signaling, Department of Molecular Biology, ULB Neuroscience Institute, Université libre de Bruxelles (ULB), Gosselies B-6041, Belgium.

Wnt signaling is key to many developmental, physiological, and disease processes in which cells seem able to discriminate between multiple Wnt ligands. This selective Wnt recognition or "decoding" capacity has remained enigmatic because Wnt/Frizzled interactions are largely incompatible with monospecific recognition. Gpr124 and Reck enable brain endothelial cells to selectively respond to Wnt7. We show that Reck binds with low micromolar affinity to the intrinsically disordered linker region of Wnt7. Availability of Reck-bound Wnt7 for Frizzled signaling relies on the interaction between Gpr124 and Dishevelled. Through polymerization, Dishevelled recruits Gpr124 and the associated Reck-bound Wnt7 into dynamic Wnt/Frizzled/Lrp5/6 signalosomes, resulting in increased local concentrations of Wnt7 available for Frizzled signaling. This work provides mechanistic insights into the Wnt decoding capacities of vertebrate cells and unravels structural determinants of the functional diversification of Wnt family members.
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http://dx.doi.org/10.1126/science.aat1178DOI Listing
August 2018

Probing Single Virus Binding Sites on Living Mammalian Cells Using AFM.

Methods Mol Biol 2018 ;1814:483-514

Louvain Institute of Biomolecular Science and Technology, Université catholique de Louvain, Louvain-la-Neuve, Belgium.

In the last years, atomic force microscopy (AFM)-based approaches have evolved into a powerful multiparametric tool that allows biological samples ranging from single receptors to membranes and tissues to be probed. Force-distance curve-based AFM (FD-based AFM) nowadays enables to image living cells at high resolution and simultaneously localize and characterize specific ligand-receptor binding events. In this chapter, we present how FD-based AFM permits to investigate virus binding to living mammalian cells and quantify the kinetic and thermodynamic parameters that describe the free-energy landscape of the single virus-receptor-mediated binding. Using a model virus, we probed the specific interaction with cells expressing its cognate receptor and measured the affinity of the interaction. Furthermore, we observed that the virus rapidly established specific multivalent interactions and found that each bond formed in sequence strengthens the attachment of the virus to the cell.
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http://dx.doi.org/10.1007/978-1-4939-8591-3_29DOI Listing
March 2019
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