Fast, high-resolution, non-destructive and quantitative characterization methods are needed to develop materials with tailored properties at the nanoscale or to understand the relationship between mechanical properties and cell physiology. This review introduces the state-of-the-art force microscope-based methods to map at high-spatial resolution the elastic and viscoelastic properties of soft materials. The experimental methods are explained in terms of the theories that enable the transformation of observables into material properties. Several applications in materials science, molecular biology and mechanobiology illustrate the scope, impact and potential of nanomechanical mapping methods.

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Nanomechanical mapping of soft materials with the atomic force microscope: methods, theory and applications.

Maintenance of lipid asymmetry across the two leaflets of the plasma membrane (PM) bilayer is a ubiquitous feature of eukaryotic cells. Loss of this asymmetry has been widely associated with cell death. However, increasing evidence points to the physiological importance of non-apoptotic, transient changes in PM asymmetry. Such transient scrambling events are associated with a range of biological functions, including intercellular communication and intracellular signaling. Thus, regulation of interleaflet lipid distribution in the PM is a broadly important but underappreciated cellular process with key physiological and structural consequences. Here, we compile the mounting evidence revealing multifaceted, functional roles of PM asymmetry and transient loss thereof. We discuss the consequences of reversible asymmetry on PM structure, biophysical properties and interleaflet coupling. We argue that despite widespread recognition of broad aspects of membrane asymmetry, its importance in cell biology demands more in-depth investigation of its features, regulation, and physiological and pathological implications.

Structural and functional consequences of reversible lipid asymmetry in living membranes.

Extracellular vesicles (EVs) contribute to several pathophysiological processes and appear as emerging targets for disease diagnosis and therapy However, successful translation from bench to bedside requires deeper understanding of EVs, in particular their diversity, composition, biogenesis and shedding mechanisms In this review, we focus on plasma membrane-derived microvesicles (MVs), far less appreciated than exosomes We integrate documented mechanisms involved in MV biogenesis and shedding, focusing on the red blood cell as a model We then provide a perspective for the relevance of plasma membrane lipid composition and biophysical properties in microvesiculation on red blood cells but also platelets, immune and nervous cells as well as tumor cells Although only a few data are available in this respect, most of them appear to converge to the idea that modulation of plasma membrane lipid content, transversal asymmetry and lateral heterogeneity in lipid domains may play a significant role in the vesiculation process We suggest that lipid domains may represent platforms for inclusion/exclusion of membrane lipids and proteins into MVs and that MVs could originate from distinct domains during physiological processes and disease evolution

Plasma Membrane Lipid Domains as Platforms for Vesicle Biogenesis and Shedding

Multivalent interactions play a critical role in a variety of biological processes on both molecular and cellular levels. We have used molecular force spectroscopy to investigate the strength of multiple parallel peptide-antibody bonds using a system that allowed us to determine the rupture forces and the number of ruptured bonds independently. In our experiments the interacting molecules were attached to the surfaces of the probe and sample of the atomic force microscope with flexible polymer tethers, and unique mechanical signature of the tethers determined the number of ruptured bonds. We show that the rupture forces increase with the number of interacting molecules and that the measured forces obey the predictions of a Markovian model for the strength of multiple parallel bonds. We also discuss the implications of our results to the interpretation of force spectroscopy measurements in multiple bond systems.

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Strength of Multiple Parallel Biological Bonds

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.

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

Red blood cells feature remarkable mechanical properties while navigating through microcirculation vessels and during spleen filtration. An unusual combination of plasma membrane and cytoskeleton physical properties allows red blood cells to undergo extensive deformation. Here we used atomic force microscopy multiparametric imaging to probe how cellular organization influences nanoscale and global mechanical properties of cells in both physiological and pathological conditions. Our data obtained in native conditions confirmed that, compared to healthy cells, cells from patients with hereditary spherocytosis are stiffer. Through vertical segmentation of the cell elasticity, we found that healthy and pathological cells display nanoscale architecture with an increasing stiffness along the direction of the applied force. By decoupling the mechanical response of the plasma membrane from its underlying cytoskeleton, we find that both components show altered properties in pathological conditions. Nanoscale multiparametric imaging also revealed lipid domains that exhibit differential mechanical properties than the bulk membrane in both healthy and pathological conditions. Thanks to correlated AFM-fluorescence imaging, we identified submicrometric sphingomyelin-enriched lipid domains of variable stiffness at the red blood cell surface. Our experiments provide novel insights into the interplay between nanoscale organization of red blood cell plasma membrane and their nanomechanical properties. Overall, this work contributes to a better understanding of the complex relationship between cellular nanoscale organization, cellular nanomechanics and how this 3D organization is altered in pathological conditions.

Nanoscale membrane architecture of healthy and pathological red blood cells.

Specific Interactions Measured by AFM on Living Cells between Peroxiredoxin-5 and TLR4: Relevance for Mechanisms of Innate Immunity

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. Reovirus endocytosis depends on junctional adhesion molecule A (JAM-A) and β1 integrin binding. Here, Koehler et al. use single-virus force spectroscopy and confocal microscopy to demonstrate a direct interaction between reovirus and β1 integrins via viral capsid protein λ2, which promotes clathrin recruitment to cell-bound reovirus.

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

Human peroxiredoxin-5 (PRDX5) is a unique redox-sensitive protein that plays a dual role in brain ischemia-reperfusion injury. While intracellular PRDX5 has been reported to act as a neuroprotective antioxidative enzyme by scavenging peroxides, once released extracellularly from necrotic brain cells, the protein aggravates neural cell death by inducing expression of proinflammatory cytokines in macrophages through activation of Toll-like receptor (TLR) 2 (TLR2) and 4 (TLR4). Although recent evidence showed that PRDX5 was able to interact directly with TLR4, little is known regarding the role of the cysteine redox state of PRDX5 on its DAMP function. To gain insights into the role of PRDX5 redox-active cysteine residues in the TLR4-dependent proinflammatory activity of the protein, we used a recombinant human PRDX5 in the disulfide (oxidized) form and a mutant version lacking the peroxidatic cysteine, as well as chemically reduced and hyperoxidized PRDX5 proteins. We first analyzed the oxidation state and oligomerization profile by Western blot, mass spectrometry, and SEC-MALS. Using ELISA, we demonstrate that the disulfide bridge between the enzymatic cysteines is required to allow improved TLR4-dependent IL-8 secretion. Moreover, single-molecule force spectroscopy experiments revealed that TLR4 alone is not sufficient to discriminate the different PRDX5 redox forms. Finally, flow cytometry binding assays show that disulfide PRDX5 has a higher propensity to bind to the surface of living TLR4-expressing cells than the mutant protein. Taken together, these results demonstrate the importance of the redox state of PRDX5 cysteine residues on TLR4-induced inflammation.

Mégane A. Poncin

Papers

Nanoscale membrane architecture of healthy and pathological red blood cells.

Specific Interactions Measured by AFM on Living Cells between Peroxiredoxin-5 and TLR4: Relevance for Mechanisms of Innate Immunity

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

Role of the Redox State of Human Peroxiredoxin-5 on Its TLR4-Activating DAMP Function