Force-distance Curve-based Atomic Force Microscopy Research Articles

Recognition Imaging of Trace E-cadherins on the Bladder Cancer Cells Surface during Epithelial-Mesenchymal Transition by Force-Distance Curve-Based AFM.

Tumor-associated epithelial-mesenchymal transition (EMT) contains a set of transitional cellular states usually judged by the EMT marker expression. E-cadherin is a down-regulated EMT epithelial marker, and the detection of E-cadherin is challenging on cancer cell surfaces in the middle and late stages of EMT. Here, the trace E-cadherins on the living bladder cancer T24 cell surface during EMT were investigated with force-distance curve-based atomic force microscopy. The results confirmed that T24 cells are still in an intermediate state and can be transferred into the mesenchymal phenotype by long-term TGF-β1 induction. During EMT, E-cadherins on the T24 cell surface gradually decreased and rarely clustered. E-cadherin is not completely missing, even at the end of EMT, but is too sparse to cluster. This work provides us with a visual understanding of the expression and distribution of trace markers during EMT and a deep comprehension of the indispensable significance of E-cadherin in cancer cells.

Revealing the Effect of Photothermal Therapy on Human Breast Cancer Cells: A Combined Study from Mechanical Properties to Membrane HSP70

Hyperthermia-induced overexpression of heat shock protein 70 (HSP70) leads to the thermoresistance of cancer cells and reduces the efficiency of photothermal therapy (PTT). In contrast, cancer cell-specific membrane-associated HSP70 has been proven to activate antitumor immune responses. The dual effect of HSP70 on cancer cells inspires us that in-depth research of membrane HSP70 (mHSP70) during PTT treatment is essential. In this work, a PTT treatment platform for human breast cancer cells (MCF-7 cells) based on a mPEG-NH2-modified polydopamine (PDA)-coated gold nanorod core-shell structure (GNR@PDA-PEG) is developed. Using the force-distance curve-based atomic force microscopy (FD-based AFM), we gain insight into the PTT-induced changes in the morphology, mechanical properties, and mHSP70 expression and distribution of individual MCF-7 cells with high-resolution at the single-cell level. PTT treatment causes pseudopod contraction of MCF-7 cells and generates a high level of intracellular reactive oxygen species, which severely disrupt the cytoskeleton, leading to a decrease in cellular mechanical properties. The adhesion maps, which are recorded by aptamer A8 functional probes using FD-based AFM, reveal that PTT treatment causes a significant upregulation of mHSP70 expression and it starts to exhibit a partial aggregation distribution on the MCF-7 cell surface. This work not only exemplifies that AFM can be a powerful tool for detecting changes in cancer cells during PTT treatment but also provides a better view for targeting mHSP70 for cancer therapy.

AFM-based force spectroscopy unravels stepwise formation of the DNA transposition complex in the widespread Tn3 family mobile genetic elements.

Transposon Tn4430 belongs to a widespread family of bacterial transposons, the Tn3 family, which plays a prevalent role in the dissemination of antibiotic resistance among pathogens. Despite recent data on the structural architecture of the transposition complex, the molecular mechanisms underlying the replicative transposition of these elements are still poorly understood. Here, we use force-distance curve-based atomic force microscopy to probe the binding of the TnpA transposase of Tn4430 to DNA molecules containing one or two transposon ends and to extract the thermodynamic and kinetic parameters of transposition complex assembly. Comparing wild-type TnpA with previously isolated deregulated TnpA mutants supports a stepwise pathway for transposition complex formation and activation during which TnpA first binds as a dimer to a single transposon end and then undergoes a structural transition that enables it to bind the second end cooperatively and to become activated for transposition catalysis, the latter step occurring at a much faster rate for the TnpA mutants. Our study thus provides an unprecedented approach to probe the dynamic of a complex DNA processing machinery at the single-particle level.

A 3D surface nanomechanical property mapping method with a magnetic-drive orthogonal cantilever probe.

Traditional force-distance curve based atomic force microscopy (FD-AFM) is limited to two-dimensional (2D) surface characterization, making the in situ mapping of three-dimensional (3D) surface nanomechanical properties (SNMP) challenging. This paper presents a multimode 3D FD-AFM based on a magnetic-drive orthogonal cantilever probe (MD-OCP) that can achieve SNMP imaging of 3D micro-nano structures with surface contour fluctuations reaching or exceeding several microns. Bending, torsion and vector tracking modes are integrated into this method for a 2D horizontal surface, 2D sidewall, and 3D surface mapping, respectively. The MD-OCP consists of a horizontal cantilever, a vertical cantilever with a protruding tip, and a magnetized bead. It can be utilized in the detection of deep trench and dense microarray units. The force analysis during 3D SNMP measurement is performed through mathematical derivation, which shows a clear relationship between effective indentation force, friction, and total tip-sample interactions. Single-point SNMP evaluation, discrete 2D SNMP imaging, and continuous omnidirectional 3D SNMP mapping of a 3D microarray unit verify the accurate and comprehensive measurement abilities of the reported method in its bending, torsion, and vector tracking modes. The experimental results demonstrate that this method can achieve excellent 3D quantitative characterization of topography and SNMP, including critical dimensions, adhesion, Young's modulus, stiffness, and energy dissipation, along a 3D device surface. This novel 3D FD-AFM technique has many potential applications in the further exploration of 3D micro-nano devices.

Stepwise Enzymatic-Dependent Mechanism of Ebola Virus Binding to Cell Surface Receptors Monitored by AFM.

Ebola virus (EBOV) is responsible for several outbreaks of hemorrhagic fever with high mortality, raising great public concern. Several cell surface receptors have been identified to mediate EBOV binding and internalization, including phosphatidylserine (PS) receptors (TIM-1) and C-type lectin receptors (DC-SIGNR). However, the role of TIM-1 during early cell surface binding remains elusive and in particular whether TIM-1 acts as a specific receptor for EBOV. Here, we used force-distance curve-based atomic force microscopy (FD-based AFM) to quantify the binding between TIM-1/DC-SIGNR and EBOV glycoprotein (GP) and observed that both receptors specifically bind to GP with high-affinity. Since TIM-1 can also directly interact with PS at the single-molecule level, we also confirmed that TIM-1 acts as dual-function receptors of EBOV. These results highlight the direct involvement of multiple high-affinity receptors in the first steps of binding to cell surfaces, thus offering new perspectives for the development of anti-EBOV therapeutic molecules.

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

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 in situ 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.

Simultaneously Measuring Force and Displacement: Calibration of Magnetic Torque Actuated Microcantilevers for Nanomechanical Mapping

Force-distance curve-based atomic force microscopy (FD-based AFM) has been extensively used to study the interactive phenomena and mechanical properties of various materials at the nanoscale. Unlike the commonly used piezodrive FD-based AFM that uses two sensors (cantilever and piezoactuator), the magnetic-drive FD-based AFM uses only one sensor of an off-resonant oscillating cantilever to measure the FD curve. However, due to inconsistent probe sensitivities induced by the force and magnetic torque, locations of the laser spot and magnetic bead need to be specified for broad modulus range nanomechanical mapping. Here, a calibration method is presented to obtain a uniform probe sensitivity induced by the force and magnetic torque. In this method, the torque-induced deflection sensitivity is accurately determined by the deflection tracking approach. Impacts of the laser spot and magnetic bead location on deflection sensitivity are investigated, and a straight protocol is developed to locate the laser spot for unifying two deflection sensitivities. The proposed calibration methods are applied in mapping nanomechanical properties of polystyrene, highly oriented pyrolytic graphite surfaces at driving frequencies of 5 kHz in air, and polydimethylsiloxane surface at 0.5 kHz in liquid environment.

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

Inflammation is a pathophysiological response of innate immunity to infection or tissue damage. This response is among others triggered by factors released by damaged or dying cells, termed damage-associated molecular pattern (DAMP) molecules that act as danger signals. DAMPs interact with pattern recognition receptors (PRRs) to contribute to the induction of inflammation. However, how released peroxiredoxins (PRDXs) are able to activate PRRs, such as Toll-like receptors (TLRs), remains elusive. Here, we used force-distance curve-based atomic force microscopy to investigate the molecular mechanisms by which extracellular human PRDX5 can activate a proinflammatory response. Single-molecule experiments demonstrated that PRDX5 binds to purified TLR4 receptors, on macrophage-differentiated THP-1 cells, and on human TLR4-transfected CHO cells. These findings suggest that extracellular PRDX5 can specifically trigger a proinflammatory response. Moreover, our work also revealed that PRDX5 binding induces a cellular mechanoresponse. Collectively, this study provides insights into the role of extracellular PRDX5 in innate immunity.

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

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.

Combining confocal and atomic force microscopy to quantify single-virus binding to mammalian cell surfaces.

Over the past five years, atomic force microscopy (AFM)-based approaches have evolved into a powerful multiparametric tool set capable of imaging the surfaces of biological samples ranging from single receptors to membranes and tissues. One of these approaches, force-distance curve-based AFM (FD-based AFM), uses a probing tip functionalized with a ligand to image living cells at high-resolution and simultaneously localize and characterize specific ligand-receptor binding events. Analyzing data from FD-based AFM experiments using appropriate probabilistic models allows quantification of the kinetic and thermodynamic parameters that describe the free-energy landscape of the ligand-receptor bond. We have recently developed an FD-based AFM approach to quantify the binding events of single enveloped viruses to surface receptors of living animal cells while simultaneously observing them by fluorescence microscopy. This approach has provided insights into the early stages of the interaction between a virus and a cell. Applied to a model virus, we probed the specific interaction with cells expressing viral cognate receptors 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 strengthened the attachment of the virus to the cell. Here we describe detailed procedures for probing the specific interactions of viruses with living cells; these procedures cover tip preparation, cell sample preparation, step-by-step FD-based AFM imaging and data analysis. Experienced microscopists should be able to master the entire set of protocols in 1 month.

Detecting Ligand-Binding Events and Free Energy Landscape while Imaging Membrane Receptors at Subnanometer Resolution.

Force-distance curve-based atomic force microscopy has emerged into a sophisticated technique for imaging cellular membranes and for detecting specific ligand-binding events of native membrane receptors. However, so far the resolution achieved has been insufficient to structurally map ligand-binding sites onto membrane proteins. Here, we introduce experimental and theoretical approaches for overcoming this limitation. To establish a structurally and functionally well-defined reference sample, we engineer a ligand-binding site to the light-driven proton pump bacteriorhodopsin of purple membrane. Functionalizing the AFM stylus with an appropriate linker-system tethering the ligand and optimizing the AFM conditions allows for imaging the engineered bacteriorhodopsin at subnanometer resolution while structurally mapping the specific ligand-receptor binding events. Improved data analysis allows reconstructing the ligand-binding free energy landscape from the experimental data, thus providing thermodynamic and kinetic insight into the ligand-binding process. The nanoscopic method introduced is generally applicable for imaging receptors in native membranes at subnanometer resolution and for systematically mapping and quantifying the free energy landscape of ligand binding.

Imaging G protein-coupled receptors while quantifying their ligand-binding free-energy landscape.

Imaging native membrane receptors and testing how they interact with ligands is of fundamental interest in the life sciences but has proven remarkably difficult to accomplish. Here, we introduce an approach that uses force-distance curve-based atomic force microscopy to simultaneously image single native G protein-coupled receptors in membranes and quantify their dynamic binding strength to native and synthetic ligands. We measured kinetic and thermodynamic parameters for individual protease-activated receptor-1 (PAR1) molecules in the absence and presence of antagonists, and these measurements enabled us to describe PAR1's ligand-binding free-energy landscape with high accuracy. Our nanoscopic method opens an avenue to directly image and characterize ligand binding of native membrane receptors.

Localizing chemical groups while imaging single native proteins by high-resolution atomic force microscopy.

Simultaneous high-resolution imaging and localization of chemical interaction sites on single native proteins is a pertinent biophysical, biochemical, and nanotechnological challenge. Such structural mapping and characterization of binding sites is of importance in understanding how proteins interact with their environment and in manipulating such interactions in a plethora of biotechnological applications. Thus far, this challenge remains to be tackled. Here, we introduce force-distance curve-based atomic force microscopy (FD-based AFM) for the high-resolution imaging of SAS-6, a protein that self-assembles into cartwheel-like structures. Using functionalized AFM tips bearing Ni(2+)-N-nitrilotriacetate groups, we locate specific interaction sites on SAS-6 at nanometer resolution and quantify the binding strength of the Ni(2+)-NTA groups to histidine residues. The FD-based AFM approach can readily be applied to image any other native protein and to locate and structurally map histidine residues. Moreover, the surface chemistry used to functionalize the AFM tip can be modified to map other chemical interaction sites.

Multiparametric imaging of biological systems by force-distance curve–based AFM

A current challenge in the life sciences is to understand how biological systems change their structural, biophysical and chemical properties to adjust functionality. Addressing this issue has been severely hampered by the lack of methods capable of imaging biosystems at high resolution while simultaneously mapping their multiple properties. Recent developments in force-distance (FD) curve-based atomic force microscopy (AFM) now enable researchers to combine (sub)molecular imaging with quantitative mapping of physical, chemical and biological interactions. Here we discuss the principles and applications of advanced FD-based AFM tools for the quantitative multiparametric characterization of complex cellular and biomolecular systems under physiological conditions.