Single-molecule force spectroscopy study of interaction between transforming growth factor beta1 and its receptor in living cells

Transforming growth factor beta1 (TGF-beta1) regulates many important cellular processes such as cell proliferation, differentiation, and apoptosis, etc. Its signaling is initiated by binding to and bringing together TGF-beta type II receptor (TbetaRII) and type I receptor (TbetaRI). However, it is not fully understood how the TGF-beta1 ligand-receptor interaction occurs in living cells and what is the molecular mechanism of the signaling complex TGF-beta1/TbetaRII/TbetaRI formation. In this study, we have investigated the interaction between TGF-beta1 and its receptors in living cells with single-molecule force spectroscopy for the first time. By positioning TGF-beta1-modified atomic force microscope (AFM) tips on the cells expressing fluorescent protein tagged TGF-beta receptors, the living-cell force measurement was realized with a combined fluorescence microscope and AFM. We found that coexpression of TbetaRI with TbetaRII enhanced the binding force of TGF-beta1 with its receptors, whereas the expressed TbetaRI itself exhibited no binding affinity to TGF-beta1. Moreover, the unbinding dynamics of TGF-beta1/TbetaRII and TGF-beta1/TbetaRI/TbetaRII were investigated with dynamic force spectroscopy under different AFM loading rates. The dissociation rate constants of TGF-beta1 with its receptors as well as other parameters characterizing their dissociation pathways were obtained. The results suggested a more stable binding of TGF-beta1 with the receptor after TbetaRI is recruited and the important contribution of TbetaRI to the signaling complex formation during TGF-beta1 signaling.     

Effect of the cell type and cell density on the binding of living cells to a silica particle: an atomic force microscope study

We used the atomic force microscope to study how the cell type and the density of cells adsorbed at a substrate can affect the adhesion between a living cell and a model drug delivery system (DDS) carrier nano-particle. We used three different anchorage-dependent cells, i.e., a living mouse fibroblast cell (L929), a living human colon cancer cell (Caco2), and a living mouse malignant melanoma cell (B16F10). For the DDS model nano-particle, we used a silica colloid. In order to correlate the adhesion force with the cell types, the growth curve of the cells were determined with a haemocytometer. The shapes of the cells at the different stages were monitored by light microscopy, and the morphology of their surfaces obtained by tapping mode atomic force microscopy.

Force measurements showed that the Caco2 cell bound little to a silica particle, regardless of the cell density. The L929 cell bound well to a silica particle for low and high cell densities. The B16F10 cell bound little to a silica particle for low cell densities, but bound well for high cell densities. AFM images showed that the L929 cell did not contain folds. The B16F10 cells, however, displayed folds in the cell surface for low cell densities, but no folds in the cell for high cell densities. As literature also reported that the Caco2 cell contains folds, these results suggested that cells with folds showed less adhesion to a silica particle than cells without folds. The presence of folds in the cell presumably decreased the number of sites on the cell that could hydrogen bond or undergo van der Waals binding with the silanol groups of the silica particle.     

Antigen binding forces of single antilysozyme Fv fragments explored by atomic force microscopy

We used atomic force microscopy (AFM) to explore the antigen binding forces of individual Fv fragments of antilysozyme antibodies (Fv). To detect single molecular recognition events, genetically engineered histidine-tagged Fv fragments were coupled onto AFM tips modified with mixed self-assembled monolayers (SAMs) of nitrilotriacetic acid- and tri(ethylene glycol)-terminated alkanethiols while lysozyme (Lyso) was covalently immobilized onto mixed SAMs of carboxyl- and hydroxyl-terminated alkanethiols. The quality of the functionalization procedure was validated using X-ray photoelectron spectroscopy (surface chemical composition), AFM imaging (surface morphology in aqueous solution), and surface plasmon resonance (SPR, specific binding in aqueous solution). AFM force-distance curves recorded at a loading rate of 5000 pN/s between Fv- and Lyso-modified surfaces yielded a distribution of unbinding forces composed of integer multiples of an elementary force quantum of approximately 50 pN that we attribute to the rupture of a single antibody-antigen pair. Injection of a solution containing free Lyso caused a dramatic reduction of adhesion probability, indicating that the measured 50 pN unbinding forces are due to the specific antibody-antigen interaction. To investigate the dynamics of the interaction, force-distance curves were recorded at various loading rates. Plots of unbinding force vs log(loading rate) revealed two distinct linear regimes with ascending slopes, indicating multiple barriers were present in the energy landscape. The kinetic off-rate constant of dissociation (k(off) approximately = 1 x 10(-3) s(-1)) obtained by extrapolating the data of the low-strength regime to zero force was in the range of the k(off) estimated by SPR.     

肿瘤细胞的原子力显微术成像及表征研究

本文简单介绍了原子力显微镜的发展史,以及原子力显微镜的工作原理、工作模式、活细胞在生理状态下的成像方式等,特别介绍了生物型原子力显微镜、高速原子力显微镜在生物学领域的研究及应用。原子力显微镜在扫描速度、扫描范围、扫描精度方面的不断改进将为肿瘤细胞学研究提供源源不断的动力。本文着重阐述了原子力显微术在肿瘤领域的研究进展,包括原子力显微镜在肿瘤细胞形貌学特性、硬度、粘弹性方面的研究现状,并对原子力显微镜在肿瘤诊断及抗肿瘤药物研发方面的应用前景进行了展望。     

Unbinding of the streptavidin-biotin complex by atomic force microscopy: a hybrid simulation study

A hybrid molecular simulation technique, which combines molecular dynamics and continuum mechanics, was used to study the single-molecule unbinding force of a streptavidin-biotin complex. The hybrid method enables atomistic simulations of unbinding events at the millisecond time scale of atomic force microscopy (AFM) experiments. The logarithmic relationship between the unbinding force of the streptavidin-biotin complex and the loading rate (the product of cantilever spring constant and pulling velocity) in AFM experiments was confirmed by hybrid simulations. The unbinding forces, cantilever and tip positions, locations of energy barriers, and unbinding pathway were analyzed. Hybrid simulation results from this work not only interpret unbinding AFM experiments but also provide detailed molecular information not available in AFM experiments.     

Probing the Nature of the Cluster Effect Observed with Synthetic Multivalent Galactosides and Peanut Agglutinin Lectin

We designed a set of multi‐galactosides with valencies ranging from one to seven and different spacer‐arm lengths. The compounds display a high structural homology for a strict assessment of multivalent phenomena. The multimers were first evaluated by an enzyme‐linked lectin assay (ELLA) toward the peanut agglutinin (PNA). The binding affinity was shown to be dependent on the spacer‐arm length, and cluster effects were observed for the galactosides bearing the shortest and the longest linkers. The latter compounds were shown to be much more potent PNA cross‐linkers in a “sandwich assay”. Dynamic light scattering (DLS) experiments also revealed the formation of soluble aggregates between heptavalent derivatives with medium or long linkers and the labeled PNA. ELLA experiments performed with valency‐controlled clusters and labeled lectins are therefore not always devoid from aggregative processes. The precise nature of the multivalent interaction observed by ELLA for the compounds bearing the shortest linkers, which are unable to form PNA aggregates, was further investigated by atomic force microscopy (AFM). The galactosides were grafted onto the tip of a cantilever and the PNA lectin onto a gold surface. Similar unbinding forces were registered when the valency of the ligands was increased, thus showing that the multimers cannot interact more strongly with PNA. Multiple binding events to the PNA were also never observed, thus confirming that a chelate binding mode does not operate with the multivalent galactosides, probably because the linkers are too short. Altogether, these results suggest that the cluster effect that operates in ELLA with the multimers is not related to additional PNA stabilizations and can be ascribed to local concentration effects that favor a dynamic turnover of the tethered galactosides in the PNA binding sites.     

Electrostatic orientation of enzymes on surfaces for ligand screening probed by force spectroscopy

In this letter, we show that electrostatic immobilization provides a simple but effective approach for the immobilization and orientation of carbonic anhydrase onto charged surfaces. The enzyme is oriented differently on oppositely charged surfaces, with the majority of active sites facing upward on a positively charged surface and downward on a negatively charged surface. An array of negatively charged microscale surface patterns within a positively charged background was prepared by microcontact printing and used as the substrate to immobilize the enzymes. This enabled the probing of the enzyme orientations on the two differently charged surface regions by force spectroscopy with the same atomic force microscopy (AFM) probe modified with a thiolated sulfonamide inhibitor. The unbinding forces between the inhibitor tip and the enzyme immobilized on the two differently charged surfaces were measured. Two control experiments, blocking of the enzyme active site with a competitive inhibitor and removal of the zinc ion from the enzyme catalytic center, were employed to distinguish between specific and nonspecific interactions and to further verify the differences in enzyme orientation. Autocorrelation analysis of the force histograms was carried out to evaluate the specific single enzyme-inhibitor interaction force.     

Analysis of subcellular surface structure, function and dynamics

Analytics of single biological cells allows quantitative investigation from a structural, functional and dynamical point of view and opens novel possibilities to an unamplified subcellular analysis. In this article, we report on three different experimental methods and their applications to single cellular systems with a subcellular sensitivity down to the single molecule level. First, the subcellular surface structure of living bacteria (Corynebacterium glutamicum) was investigated with atomic force microscopy (AFM) at the resolution of individual surface layer (S-layer) proteins; discrimination of bacterial strains that lack the expression of hexagonally packed surface layer proteins was possible. Second, quantitative measurement of individual recognition events of membrane-bound receptors on living B-cells was achieved in single cell manipulation and probing experiments with optical tweezers (OT) force spectroscopy. And third, intracellular dynamics of translocating photoactivatable GFP in plant protoplasts (Nicotiana tabacum BY-2) was quantitatively monitored by two-photon laser scanning microscopy (2PLSM).     

Simple test system for single molecule recognition force microscopy

We have established an easy-to-use test system for detecting receptor-ligand interactions on the single molecule level using atomic force microscopy (AFM). For this, avidin-biotin, probably the best characterized receptor-ligand pair, was chosen. AFM sensors were prepared containing tethered biotin molecules at sufficiently low surface concentrations appropriate for single molecule studies. A biotin tether, consisting of a 6 nm poly(ethylene glycol) (PEG) chain and a functional succinimide group at the other end, was newly synthesized and covalently coupled to amine-functionalized AFM tips. In particular, PEG₈₀₀ diamine was glutarylated, the mono-adduct NH₂-PEG-COOH was isolated by ion exchange chromatography and reacted with biotin succinimidylester to give biotin-PEG-COOH which was then activated as N-hydroxysuccinimide (NHS) ester to give the biotin-PEG-NHS conjugate which was coupled to the aminofunctionalized AFM tip. The motional freedom provided by PEG allows for free rotation of the biotin molecule on the AFM sensor and for specific binding to avidin which had been adsorbed to mica surfaces via electrostatic interactions. Specific avidin-biotin recognition events were discriminated from nonspecific tip-mica adhesion by their typical unbinding force (∼40 pN at 1.4 nN/s loading rate), unbinding length (<13 nm), the characteristic nonlinear force-distance relation of the PEG linker, and by specific block with excess of free d-biotin. The convenience of the test system allowed to evaluate, and compare, different methods and conditions of tip aminofunctionalization with respect to specific binding and nonspecific adhesion. It is concluded that this system is well suited as calibration or start-up kit for single molecule recognition force microscopy.     

Functionalized self-assembled monolayers for measuring single molecule lectin carbohydrate interactions

The specific interactions between sugar-binding proteins (lectins) and their complementary carbohydrates mediate several complex biological functions. There is a great deal of interest in uncovering the molecular basis of these interactions. In this study, we demonstrate the use of an efficient one-step amination reaction strategy to fabricate carbohydrate arrays based on mixed self-assembled monolayers. These allow specific lectin carbohydrate interactions to be interrogated at the single molecule level via AFM. The force required to directly rupture the multivalent bonds between Concanavalin A (Con A) and mannose were subsequently determined by chemical force microscopy. The mixed self-assembled monolayer provides a versatile platform with active groups to attach a 1-amino-1-deoxy sugar or a protein (Con A) while minimizing non-specific adhesion enabling quick and reliable detection of rupture forces. By altering the pH of the environment, the aggregation state of Con A was regulated, resulting in different dominant rupture forces, corresponding to di-, tri- and multiple unbinding events. We estimate the value of the rupture force for a single Con A-mannose bond to be 95 ± 10 pN. The rupture force is consistent even when the positions of the binding molecules are switched. We show that this synthesis strategy in conjunction with a mixed SAM allows determination of single molecules bond with high specificity, and may be used to investigate lectin carbohydrate interactions in the form of carbohydrate arrays as well as lectin arrays.     

Molecular Basis of the Dynamic Strength of the Sialyl Lewis X—Selectin Interaction

The selectins are Ca²⁺‐dependent cell adhesion molecules that facilitate the initial attachment of leukocytes to the vascular endothelium by binding to a carbohydrate moiety as exemplified by the tetrasaccharide, sialyl Lewis X (sLeX). An important property of the selectin‐sLeX interaction is its ability to withstand the hydrodynamic force of the blood flow. Herein, we used single‐molecule dynamic force spectroscopy (DFS) to identify the molecular determinants within sLeX that give rise to the dynamic properties of the selectin/sLeX interaction. Our atomic force microscopy (AFM) measurements revealed that the unbinding of the selectin/sLeX complexes involves overcoming at least two activation barriers. The inner barrier, which determines the dynamic response of the complex at high forces, is governed by the interaction between the Fuc residue of sLeX and a Ca²⁺ ion chelated to the lectin domain of the selectin molecule, whereas the outer activation barrier can be attributed to interactions involving the sialic acid residue of sLeX. Due to their steep inner activation barriers, the selectin‐sLeX complexes are less sensitive to high pulling forces. Hence, besides its contribution to the bond energy, the Ca²⁺ ion also grants the selectin–sLeX complexes a tensile strength that is crucial for the selectin‐mediated rolling of leukocytes.     

Detection of transglucosidase-catalyzed polysaccharide synthesis on a surface in real time using surface plasmon resonance spectroscopy

Biological systems that involve enzyme catalysis at surfaces, particularly strategically important ones that involve insoluble substrates/products such as the cell wall and the starch granule, require analyses beyond classical solution state enzymology. Using a model system, we have demonstrated the real-time measurement of transglucosidase activity on a surface using surface plasmon resonance (SPR) spectroscopy. We monitored the extension of a (partially carboxymethylated) dextran surface with alternansucrase and sucrose as a glycosyl donor. Conditions were used where surface polymer synthesis rates were a function of enzyme concentration and proportional to the extent of enzyme binding to the surface. A method to determine the turnover number of the enzyme on the surface was also developed. The presence of a new amorphous polysaccharide was observed optically, detected by lectin binding and imaged by atomic force microscopy. This surface method will have utility in a wide range of carbohydrate enzyme systems including screens.     

Atomic force microscopy of microvillous cell surface dynamics at fixed and living alveolar type II cells

Scanning probe techniques enable direct imaging of morphology changes associated with cellular processes at life specimen. Here, glutaraldehyde-fixed and living alveolar type II (ATII) cells were investigated by atomic force microscopy (AFM), and the obtained topographical data were correlated with results obtained by scanning electron microscopy (SEM) and confocal microscopy (CM). We show that low-force contact mode AFM at glutaraldehyde-fixed cells provides complementary results to SEM and CM. Both AFM and SEM images reveal fine structures at the surface of fixed cells, which indicate microvilli protrusions. If ATII cells were treated with Ca²⁺ channel modulators known to induce massive endocytosis, changes of the cell surface topography became evident by the depletion of microvilli. Low force contact mode AFM imaging at fixed ATII cells revealed a significant reduction of the surface roughness for capsazepine and 2-aminoethoxydiphenyl-borate (CPZ/2-APB)-treated cells compared to untreated control cells (Rc of 99.7 ± 6.8 nm vs. Rc of 71.9 ± 4.6 nm for N = 22), which was confirmed via SEM studies. CM of microvilli marker protein Ezrin revealed a cytoplasmic localization of Ezrin in CPZ/2-APB-treated cells, whereas a submembranous Ezrin localization was observed in control cells. Furthermore, in situ AFM investigations at living ATII cells using low force contact mode imaging revealed an apparent decrease in cell height of 17% during stimulation experiments. We conclude that a dynamic reorganization of the microvillous cell surface occurs in ATII cells at conditions of stimulated endocytosis.     

Visualized investigation of yeast transformation induced with Li+ and polyethylene glycol

The effects of Li⁺ and polyethylene glycol (PEG) on the genetic transformation of Saccharomyces cerevisiae were investigated by using fluorescence microscopy (FM) to visualize the binding of plasmid DNA labeled with YOYO-1 to the surface of yeast cells, scanning electron microscopy (SEM) and atomic force microscopy (AFM) to image the change in surface topography of yeast cells, coupled with transformation frequency experiments. The results showed that under the same conditions, the transformation frequencies of yeast protoplasts were much higher than those of intact yeast cells. PEG was absolutely required for the binding of DNA to the surface of intact yeast cells or yeast protoplasts, and had no effect on the surface topography of intact yeast cells or yeast protoplasts. In the presence of PEG, Li⁺ could greatly enhance the binding of plasmid DNA to the surface of intact yeast cells, increase their transformation frequency, and affect their surface topography. On the other hand, no effect on the DNA binding to the surface of protoplasts and no increase in the number of transformants and no surface topography changes were found upon the treatment with Li⁺ to protoplasts. In the present work, the effects of Li⁺ and PEG on yeast genetic transformation were directly visualized, rather than those deduced from the results of transformation frequencies. These results indicate that cell wall might be a barrier for the uptake of plasmid DNA. Li⁺ could increase the permeability of yeast cell wall, then increase the exposed sites of DNA binding on intact yeast cells. The main role of PEG was to induce DNA binding to cell surface.     

Parameters affecting the adhesion strength between a living cell and a colloid probe when measured by the atomic force microscope

In this study, we used the colloid probe atomic force microscopy (AFM) technique to investigate the adhesion force between a living cell and a silica colloid particle in a Leibovitz's L-15 medium (L-15). The L-15 liquid maintained the pharmaceutical conditions necessary to keep the cells alive in the outside environment during the AFM experiment. The force curves in such a system showed a steric repulsion in the compression force curve, due to the compression of the cells by the colloid probe, and an adhesion force in the decompression force curve, due to binding events between the cell and the probe. We also investigated for the first time how the position on the cell surface, the strength of the pushing force, and the residence time of the probe at the cell surface individually affected the adhesion force between a living cell and a 6.84 μm diameter silica colloid particle in L-15. The position of measuring the force on the cell surface was seen not to affect the value of the maximum adhesion force. The loading force was also seen not to notably affect the value of the maximum adhesion force, if it was small enough not to pierce and damage the cell. The residence time of the probe at the cell surface, however, clearly affected the adhesion force, where a longer residence time gave a larger maximum force. From these results, we could conclude that the AFM force measurements should be made using a loading force small enough not to damage the cell and a fixed residence time, when comparing results of different systems.     

Single-photon atomic force microscopy

In the last few years, an array of novel technologies, especially the big family of scanning probe microscopy, now often integrated with other powerful imaging tools such as laser confocal microscopy and total internal reflection fluorescence microscopy, have been widely applied in the investigation of biomolecular interactions and dynamics. But it is still a great challenge to directly monitor the dynamics of biomolecular interactions with high spatial and temporal resolution in living cells. An innovative method termed “single-photon atomic force microscopy” (SP-AFM), superior to existing techniques in tracing biomolecular interactions and dynamics in vivo, was proposed on the basis of the combination of atomic force microscopy with the technologies of carbon nanotubes and single-photon detection. As a unique tool, SP-AFM, capable of simultaneous topography imaging and molecular identification at the subnanometer level by synchronous acquisitions and analyses of the surface topography and fluorescent optical signals while scanning the sample, could play a very important role in exploring biomolecular interactions and dynamics in living cells or in a complicated biomolecular background.     

Dynamic force spectroscopy of the specific interaction between the PDZ domain and its recognition peptides

To characterize the molecular basis of specific interactions of PDZ proteins, dynamic force spectroscopy (DFS) for the PDZ protein Tax-interacting protein-1 (TIP-1) and its recognition peptide (PDZ-pep) derived from beta-catenin was performed using an atomic force microscope (AFM), together with measurement of thermodynamic and kinetic parameters using surface plasmon resonance (SPR). The unbinding force of this pair was measured under different conditions of AFM tip-retraction velocity. The relationship between the unbinding force and the logarithmic force-loading rate, that is, the dynamic force spectrum, exhibited two different rate regimes, for each of which the forces increased linearly with the force-loading rate. On the basis of the theoretical treatment of the Bell-Evans model, the positions of two different activation barriers in the reaction coordinate and dissociation rate constants in each barrier were evaluated from slopes and x-intercepts of the two linear regimes (first barrier: 0.04 nm and 1.10 x 10 s(-1); second barrier: 0.21 nm and 2.77 x 10(-2) s(-1), respectively). Although two-step unbinding kinetics between TIP-1 and PDZ-pep was suggested from the DFS analysis, SPR results showed single-step dissociation kinetics with a rate constant of 2.89 x 10(-1) s(-1). Different shapes of the free energy profile of the unbinding process were deduced from each result of DFS and SPR. The reason for such topographic differences in the energy landscape is discussed in relation to the differences in the pathways of forced unbinding and spontaneous dissociation.     

Atomic force microscopy studies of ganglioside GM1alpha in dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine mixed monolayers and hybrid bilayers

The membrane states of the alpha-series ganglioside GM1alpha in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) mixed monolayers and hybrid bilayers were investigated using atomic force microscopy (AFM). The AFM image for the GM1alpha/DOPC/DPPC ternary monolayers showed the formation of GM1alpha-raft in the DOPC matrix. As increase of the surface pressure, GM1alpha are condensed in DPPC-rich domains; long and slender GM1alpha-rafts are separated from the DPPC-rich domains into the DOPC matrix. The GM1alpha/DOPC/DPPC ternary monolayers were deposited on mica coated with the first layer (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine: DPPE) using the Langmuir-Schaeffer technique. The AFM image for the hybrid bilayers showed that same molecules were heterogeneously concentrated according to increase of the surface pressure to form GM1alpha-raft, DPPC-rich domain and DOPC matrix, being in agreement with the observation on the monolayer experiment. The found phenomenon implies that a binding of lectin to GM1alpha causes the increase of the surface pressure, the localization of GM1alpha and the succeeding formation of the raft as a first step of a specific signal transduction.     

Investigation of the interaction between a bivalent aptamer and thrombin by AFM

Aptamers are a new class of molecular probes for protein recognition, detection, and inhibition. Multivalent aptamer-protein binding through aptamer assembly has been currently developed as an effective way to achieve higher protein affinity and selectivity. In this study, the specific interaction between bivalent aptamer Bi-8S and thrombin has been measured directly and quantitatively by atomic force microscopy to investigate the unbinding dynamics and dissociation energy landscape of the multivalent interaction. Bivalent aptamer Bi-8S contains thrombin's two aptamers, 15apt and 27apt, which are linked by eight spacer phosphoramidites. The results revealed the sequential dissociation of the two aptamers. Moreover, the dynamic force spectroscopy data revealed that the 27apt's binding to the thrombin remains largely unaffected by the eight-spacer phosphoramidites within Bi-8S. In contrast, the eight-spacer phosphoramidites stabilized the 15apt-thrombin binding.