Probing living cell dynamics and molecular interactions using atomic force microscopy

Abstract

Atomic force microscopy (AFM) has emerged as a powerful tool for studying biological interactions at the single-molecule level, offering unparalleled insights into receptor-ligand dynamics on living cells. This review discusses key developments in the application of AFM, highlighting its ability to capture nanomechanical properties of cellular surfaces and probe dynamic interactions, such as virus-host binding. AFM's versatility in measuring mechanical forces and mapping molecular interactions in near-physiological conditions is explored. The review also emphasizes how AFM provides critical insights into cell surface organization, receptor functionality, and viral entry mechanisms, advancing the understanding of cellular and molecular processes.

Keywords: Atomic force microscopy; Dynamic force spectroscopy; Interactions; Ligand-receptor; Single-molecule force spectroscopy.

Fig. 1

Main components of an AFM. The piezoelectric scanner enables precise sample movements in all three dimensions (x, y, z). The AFM cantilever with a sharp tip interacts with the sample surface. At the same time, a laser beam reflects off the back of the cantilever onto a photodiode, which detects deflections caused by surface interactions. A computer controller and feedback system regulate the scanning process and adjust the sample position depending on the setpoint. Created with BioRender.com

Fig. 2

AFM force spectroscopy overview. A Schematic representation of the AFM tip’s approach and retraction from the sample surface, highlighting the key interaction stages. B Force–time (FT) curve illustrating the force exerted by the AFM tip over time during the approach and retraction phases, with numbered labels corresponding to the stages in panel A. C Force-distance (FD) curve displaying the same interaction, where the force is plotted against the tip-sample distance. As the cantilever approaches the sample (label 1), the force remains at baseline. Upon contact (label 2), upward deflection occurs, followed by a predefined force setpoint (label 3) to assess the sample’s mechanical properties. During retraction (label 4), if an interaction is present, downward bending of the cantilever is observed, leading to a characteristic signal until the interaction breaks (label 5). If no interaction occurs, the force returns directly to baseline. Created with BioRender.com

Fig. 3

Principle of AFM mapping and tip functionalization strategies. A, B Schematic representation of AFM force mapping, where the AFM tip undergoes approach-retraction cycles for each pixel across the sample surface. The tip movement can follow either a linear (rectangular) path (A) or an oscillating (sinusoidal) waveform (B). C Various AFM tip functionalization strategies are shown. Left: Chemical functionalization of tips for chemical force microscopy, such as gold tips modified with self-assembled monolayers of alkanethiols. Right: Biomolecule attachment using flexible linkers like poly(ethylene glycol) (PEG), offering improved mobility for molecular interactions. Created with BioRender.com

Fig. 4

Dynamic binding of glucagon to the glucagon receptor (GCGR) investigated using force-distance (FD)–based AFM. A Schematic of the AFM tip functionalization with glucagon, covalently immobilized via a C-terminal cysteine using a heterobifunctional linker. B Topographical overview of apo-GCGRs before (top) and after (bottom) 30 min of incubation with glucagon. Zoomed topographs and cross-sections of GCGRs extracted from black (inactive) and red (active) circles show a clear increase in receptor height upon glucagon binding. C Height distribution of glucagon-GCGR complexes acquired in situ with glucagon-functionalized AFM tips, highlighting the transition between inactive and active receptor states. D Representative force-distance curves showing specific binding, non-specific interactions, and no adhesion events. E Force vs. loading rate plots for glucagon-GCGR interactions (n = 832), with Friddle-Noy-De Yoreo fits (dashed lines) reconstructing the ligand-receptor binding free-energy landscape for the two distinct binding states. These data reveal stepwise receptor activation and the role of mechanical forces in GCGR signaling. Adapted from Lo Giudice et al. (2020)

Fig. 5

Probing cholesterol-enriched domains in breast cancer cells using FD-AFM with θ-toxin functionalized tips. A Schematic of AFM tip functionalization with the θ-toxin via a sortase A–mediated reaction. B θ-toxin functionalized AFM tip probing plasma membrane cholesterol. Specific unbinding events (F > 100 pN) indicate cholesterol-enriched domains, while areas lacking cholesterol show minimal adhesion (F < 30 pN). C–F Co-cultured MCF10A (benign, GFP-labeled) and MCF10CA1a (malignant, unlabeled) cells. D FD-AFM height image, E adhesion map showing higher cholesterol in MCF10CA1a, and F Young’s modulus map showing higher stiffness in MCF10A. Adapted from Dumitru et al. (2020)

Fig. 6

Integration of AFM with confocal microscopy for studying virus-cell interactions under physiological conditions. A Schematic of the experimental setup combining AFM with optical microscopy in a temperature- and gas-controlled environmental chamber. B Force–time curves recorded during the interaction between rabies viruses pseudotyped with EnvA proteins (attached to the AFM tip) and MDCK cells expressing TVA receptors. The graph highlights multiple rupture events (blue, green, and red), indicative of cooperative virus-receptor binding interactions. C Imaging of mixed cultures of wild-type MDCK cells and MDCK–TVA cells expressing fluorescent proteins (red). D FD-based AFM height image of cells in the dashed region shown in panel C, displaying the surface topography of the cells. E Corresponding adhesion map, generated through force mapping, showing the spatial distribution of virus-cell binding events. Adapted from Alsteens et al. (2017)

Fig. 7

AFM as a sensor to study intracellular signaling and virus endocytosis. A–D PRDX5-TLR4 interaction studied using FD-based AFM on living THP-1 cells. A Schematic of AFM setup with PRDX5-functionalized tip probing cells under physiological conditions. B Phase-contrast image of THP-1 cells with AFM tip above (shadow visible). Inset: Height image in dashed square. C Adhesion channel image showing PRDX5-TLR4 binding. D Force vs piezo-distance curve with stiffness extracted from the contact region, showing time-dependent stiffness increase due to PRDX5 binding. E–G Reovirus early-endocytosis visualized with FluidFM and confocal microscopy. E Fluorescent T3SA + reovirus nanoparticles trapped by FluidFM probe and brought into contact with clathrin-BFP CHO-JAM-A cells. (F) Time-lapse images showing clathrin (green) recruitment to reovirus (red) contact site. G Clathrin recruitment over time, inhibited by integrin blockers (black, red) or Neu5Ac, but present without blockers (green). Adapted from Knoops et al. (2018) and Koehler et al. (2021a , b)