Recording force events of single quantum-dot endocytosis

We applied force spectroscopy based on atomic force microscope (AFM) to demonstrate the possibility of measuring the interaction force between single quantum-dots (QDs) and living cells at single particle level under native conditions. In the force-distance cycle, we recorded the events of cellular uptake of single QDs and single QD detachment from the cell.     

Nanosensing of Fcγ receptors on macrophages

Determining the distribution of specific binding sites on biological samples with high spatial accuracy (in the order of several nanometer) is an important challenge in many fields of biological science. Combination of high-resolution atomic force microscope (AFM) topography imaging with single-molecule force spectroscopy provides a unique possibility for the detection of specific molecular recognition events. The identification and localization of specific receptor binding sites on complex heterogeneous biosurfaces such as cells and membranes are of particular interest in this context. Simultaneous topography and recognition imaging was used to unravel the nanolandscape of cells of the immune system such as macrophages. The most studied phagocytic receptors include the Fc receptors that bind to the Fc portion of immunoglobulins. Here, nanomapping of FcγRs (Fc receptors for immunoglobulin G (IgG)) was performed on fixed J774.A1 mouse macrophage cell surfaces with magnetically coated AFM tips functionalized with Fc fragments of mouse IgG via long and flexible poly(ethylene glycol) linkers. Because of possible AFM tip engulfment on living macrophages, appropriate cell fixation procedure leaving the binding activity of FcγRs practically intact was elaborated. The recognition maps revealed prominent spots (microdomains) more or less homogeneously distributed on the macrophage surface with the sizes from 4 to 300 nm. Typical recognition image contained about ∼4% of large clusters (>200 nm), which were surrounded by a massive number (∼50%) of small-size (4–30 nm) and the rest by middle-size (50, 150 nm) domains. These spots were detected from the decrease of oscillation amplitude during specific binding between Fc-coated tip and FcγRs on macrophage surfaces. In addition, the effect of osmotic swelling on the topographical landscape of macrophage surfaces and on the reorganization of FcγRs was investigated.     

运用功能化修饰的碳纳米管针尖测量配体-受体的作用力

电弧法自制碳纳米管原子力显微镜针尖,对其末端进行功能化修饰,然后测量配体-受体之间的作用力。运用没有功能化修饰的碳纳米管针尖与修饰有亲和素分子的基底进行接触测量时,没有粘滞力出现;而运用末端修饰生物素分子的碳纳米管针尖测量时,有粘滞力产生。功能化的碳纳米管针尖直接测得的粘滞力均大约200pN,此值符合一对配体生物素和受体亲和素之间的作用力。这一结果很难用传统的针尖获得,功能化修饰的碳纳米管针尖能够克服传统针尖在力测量中的局限,在生物学和化学领域有着广泛的应用前景。     

原子力显微镜-荧光显微镜联用技术在活细胞单分子检测中的应用

原子力显微镜(Atomic force microscopy,AFM)及荧光显微镜(Fluorescence microscopy,FM)是目前活细胞单分子分析检测中最常用的两种工具.结合两种显微镜的优势,发展高时空分辨、多功能的AFM-FM联用技术成为近年该领域的研究热点.本文简述了AFM单分子力谱和FM单分子荧光成像的原理,总结了AFM-FM联用系统在仪器研制方面的发展概况,并结合本课题组在应用AFM-FM联用技术研究细胞膜上配受体相互作用等方面的工作,介绍了其在活细胞单分子检测中的应用进展.     

Nano-mechanical methods in biochemistry using atomic force microscopy

The atomic force microscope has been extensively used not only to image nanometer-sized biological samples but also to measure their mechanical properties by using the force curve mode of the instrument. When the analysis based on the Hertz model of indentation is applied to the approach part of the force curve, one obtains information on the stiffness of the sample in terms of Young's modulus. Mapping of local stiffness over a single living cell is possible by this method. The retraction part of the force curve provides information on the adhesive interaction between the sample and the AFM tip. It is possible to functionalize the AFM tip with specific ligands so that one can target the adhesive interaction to specific pairs of ligands and receptors. The presence of specific receptors on the living cell surface has been mapped by this method. The force to break the co-operative 3D structure of globular proteins or to separate a double stranded DNA into single strands has been measured. Extension of the method for harvesting functional molecules from the cytosol or the cell surface for biochemical analysis has been reported. There is a need for the development of biochemical nano-analysis based on AFM technology.     

Mechanostability of the Single‐Electron‐Transfer Complexes of Anabaena Ferredoxin–NADP+ Reductase

The complexes formed between the flavoenzyme ferredoxin–NADP⁺ reductase (FNR; NADP⁺=nicotinamide adenine dinucleotide phosphate) and its redox protein partners, ferredoxin (Fd) and flavodoxin (Fld), have been analysed by using dynamic force spectroscopy through AFM. A strategy is developed to immobilise proteins on a substrate and AFM tip to optimise the recognition ability. The differences in the recognition efficiency regarding a random attachment procedure, together with nanomechanical results, show two binding models for these systems. The interaction of the reductase with the natural electron donor, Fd, is threefold stronger and its lifetime is longer and more specific than that with the substitute under iron‐deficient conditions, Fld. The higher bond probability and two possible dissociation pathways in Fld binding to FNR are probably due to the nature of this complex, which is closer to a dynamic ensemble model. This is in contrast with the one‐step dissociation kinetics that has been observed and a specific interaction described for the FNR:Fd complex.     

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.     

Atomic force microscopy demonstrates that disulfide bridges are required for clustering of the yeast cell wall integrity sensor Wsc1

In yeasts, cell surface stresses are detected by a family of plasma membrane sensors. Among these, Wsc1 contains an extracellular cysteine-rich domain (CRD), which mediates sensor clustering and is believed to anchor the sensor in the cell wall. Although the formation of Wsc1 clusters and their interaction with the intracellular pathway components are important for proper stress signaling, the molecular mechanisms underlying clustering remain poorly understood. Here, we used the combination of single-molecule atomic force microscopy (AFM) with genetic manipulations to demonstrate that Wsc1 clustering involves disulfide bridges of the CRD. Using AFM tips carrying nitrilotriacetate groups, we mapped the distribution of individual His-tagged sensors on living yeast cells. While Wsc1 formed nanoscale clusters on native cells, clustering was no longer observed after treatment with the reducing agent dithiothreitol (DTT), indicating that intra- or intermolecular disulfide bridges are required for clustering. Moreover, DTT treatment resulted in a significant increase in cell surface roughness, suggesting that disulfide bridges between other cell-wall proteins are crucial for proper cell surface topology. The remarkable sensor properties unravelled here may well apply to other sensors and receptors with cysteine-rich domains throughout biology. Our combined method of AFM with genetic manipulations offers great prospects to explore the mechanisms underlying the clustering of cell surface proteins.     

Collective and single-molecule interactions of alpha5beta1 integrins

A novel biomimetic system was used to study collective and single-molecule interactions of the alpha5beta1 receptor-GRGDSP ligand system with an atomic force microscope (AFM). Bioartificial membranes, which display peptides that mimic the cell adhesion domain of the extracellular matrix protein fibronectin, are constructed from peptide-amphiphiles. The interaction measured with the immobilized alpha5beta1 integrins and GRGDSP peptide-amphiphiles is specifically related to the integrin-peptide binding. It is affected by divalent cations in a way that accurately mimics the adhesion function of the alpha5beta1 receptor. The recognition of the immobilized receptor was significantly increased for a surface that presented both the primary recognition site (GRGDSP) and the synergy site (PHSRN) compared to the adhesion measured with surfaces that displayed only the GRGDSP peptide. At the collective level, the separation process of the receptor-ligand pairs is a combination of multiple unbinding and stretching events that can accurately be described by the wormlike chain (WLC) model of polymer elasticity. In contrast, stretching was not observed at the single-molecule level. The dissociation of single alpha5beta1-GRGDSP pairs under loading rates of 1-305 nN/s revealed the presence of two activation energy barriers in the unbinding process. The high-strength regime above 59 nN/s maps the inner barrier at a distance of 0.09 nm along the direction of the force. Below 59 nN/s a low-strength regime appears with an outer barrier at 2.77 nm and a much slower transition rate that defines the dissociation rate (off-rate) in the absence of force (k(off) degrees = 0.015 s(-1)).     

采用超低场生物力谱技术研究核酸适配体与凝血酶蛋白的相互作用

蛋白质和核酸是构成生命体最为重要的两类生物大分子,它们之间的相互作用是分子生物学研究的中心问题之一,也是许多生命活动的重要组成部分。本文基于一种全新的超低场生物力谱技术,以凝血酶蛋白为研究对象,考察了凝血酶与其对应的核酸适配体之间的相互作用。结果表明,凝血酶蛋白与核酸适配体之间的结合力大小约为80 p N。同时,在分子水平上获得了凝血酶蛋白与核酸适配体之间的解离动力学信息。     

Oriented binding of the His6-tagged carboxyl-tail of the L-type Ca2+ channel alpha1-subunit to a new NTA-functionalized self-assembled monolayer

Oriented stable binding of functional proteins on surfaces is of fundamental interest for receptor/ligand studies in atomic force microscopy (AFM) and surface plasmon resonance (SPR) experiments. Here we have chosen the His6-tagged carboxyl-tail (C-tail) of the alpha1c-subunit of the L-type Ca2+ channel and calmodulin (CaM) as its cognitive partner as a model system to develop a new functional surface. Covalently attached self-assembled monolayers on ultraflat gold containing NTA-thiols to which the His6-tagged C-tail was bound and thiols with triethylene-glycol groups as matrix-thiols represented the system of choice. The topography of this surface was characterized using AFM; its ability to bind C-tail proteins oriented and stable was confirmed by SPR measurements and by complementary force spectroscopy experiments with a CaM4-construct covalently attached to the tip. The developed anchoring strategy can now be used to study receptor/ligand interactions in general applying force spectroscopy and SPR on His6-tagged proteins oriented immobilized onto this new NTA-functionalized self-assembled monolayer.     

Investigating the specific interactions between carbonic anhydrase and a sulfonamide inhibitor by single-molecule force spectroscopy

In this communication, we report on the interaction landscape of an active site-specific enzyme-inhibitor complex by single-molecule force spectroscopy. Electrostatic immobilization was employed to orient a carbonic anhydrase enzyme on a positively charged surface so its active site is pointing upward. This approach to immobilization effectively increases the number of specific interactions measured between the zinc ion of the active site on carbonic anhydrase and a sulfonamide inhibitor tethered to an atomic force microscope (AFM) probe. Further, it reduces the time required for data collection and thereby minimizes the possible mechanical damage to the probe and contamination of the enzyme surface. The rupture force measured at various loading rates is interpreted in terms of a single energy barrier for the carbonic anhydrase enzyme-sulfonamide inhibitor complex from which the kinetic and thermodynamic parameters were estimated on the basis of microscopic models and were compared to the Bell-Evans model. The dissociation rate for the enzyme-inhibitor complex was found to be significantly faster (~35 times) than the natural spontaneous dissociation rate.     

Reversible Control over Molecular Recognition in Surface‐Bound Photoswitchable Hydrogen‐Bonding Receptors: Towards Read–Write–Erase Molecular Printboards

The synthesis of an anthracene‐bearing photoactive barbituric acid receptor and its subsequent grafting onto azide‐terminated alkanethiol/Au self‐assembled monolayers by using an Cu^I‐catalyzed azide–alkyne reaction is reported. Monolayer characterization using contact‐angle measurements, electrochemistry, and spectroscopic ellipsometry indicate that the monolayer conversion is fast and complete. Irradiation of the receptor leads to photodimerization of the anthracenes, which induces the open‐to‐closed gating of the receptor by blocking access to the binding site. The process is thermally reversible, and polarization‐modulated IR reflection–absorption spectroscopy indicates that photochemical closure and thermal opening of the surface‐bound receptors occur in 70 and 100 % conversion, respectively. Affinity of the open and closed surface‐bound receptor was characterized by using force spectroscopy with a barbituric‐acid‐modified atomic force microscope tip.     

Activation of G-protein-coupled receptors in cell-derived plasma membranes supported on porous beads

G-protein-coupled receptors (GPCRs) are ubiquitous mediators of signal transduction across cell membranes and constitute a very important class of therapeutic targets. In order to study the complex biochemical signaling network coupling to the intracellular side of GPCRs, it is necessary to engineer and control the downstream signaling components, which is difficult to realize in living cells. We have developed a bioanalytical platform enabling the study of GPCRs in their native membrane transferred inside-out from live cells to lectin-coated beads, with both membrane sides of the receptor being accessible for molecular interactions. Using heterologously expressed adenosine A(2A) receptor carrying a yellow fluorescent protein, we showed that the tethered membranes comprised fully functional receptors in terms of ligand and G protein binding. The interactions between the different signaling partners during the formation and subsequent dissociation of the ternary signaling complex on single beads could be observed in real time using multicolor fluorescence microscopy. This approach of tethering inside-out native membranes accessible from both sides is straightforward and readily applied to other transmembrane proteins. It represents a generic platform suitable for ensemble as well as single-molecule measurements to investigate signaling processes at plasma membranes.     

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.     

Force measurements for membrane protein manipulation

The force curve measurement mode of the atomic force microscope (AFM) enables us to measure hitherto unobservable mechanical properties of nanometer sized biological specimens. By applying this mode, we attempted to conduct such mechanical manipulations of membrane proteins as: (1) measurement of the separation force between a membrane bound receptor and a covalently cross-linked ligand molecule on the AFM tip; and (2) extraction of membrane proteins after harnessing them on a modified tip with covalent cross-linkers. Since the limiting tensile force of the covalent system used in our experiment was a crucial factor for successful manipulations, we first estimated the force to terminate the covalent cross-linking system at the single molecular level to be 1.6–1.7 nN, based on our previous data. The method was then applied to measure the force required to separate α₂-macroglobulin (α₂-M) from its receptor on the cell membrane using an AFM tip coated with the receptor binding form of the protein. From a bimodal distribution of rupture force, we obtained an average value of 120 pN as the force to separate a non-covalent association of α₂-M with its receptor. When modified tips with covalent cross-linkers aimed at amino groups on the cell surface were used, distribution of the rupture force shifted toward higher values, with a peak in the histogram ≈400–500 pN. Since the force to sever covalent cross-linking system was 1.6–1.7 nN, the observed force was ascribed to the force required to extract membrane proteins from the cell membrane after covalent bond formation.     

Reverse engineering of an affinity-switchable molecular interaction characterized by atomic force microscopy single-molecule force spectroscopy

Tunable and switchable interaction between molecules is a key for regulation and control of cellular processes. The translation of the underlying physicochemical principles to synthetic and switchable functional entities and molecules that can mimic the corresponding molecular functions is called reverse molecular engineering. We quantitatively investigated autoinducer-regulated DNA-protein interaction in bacterial gene regulation processes with single atomic force microscopy (AFM) molecule force spectroscopy in vitro, and developed an artificial bistable molecular host-guest system that can be controlled and regulated by external signals (UV light exposure and thermal energy). The intermolecular binding functionality (affinity) and its reproducible and reversible switching has been proven by AFM force spectroscopy at the single-molecule level. This affinity-tunable optomechanical switch will allow novel applications with respect to molecular manipulation, nanoscale rewritable molecular memories, and/or artificial ion channels, which will serve for the controlled transport and release of ions and neutral compounds in the future.     

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。     

Direct measurement of recognition forces between proteins and membrane receptors

The interactions between the protein, cholera toxin B subunit attached to an atomic force microscope, AFM, cantilever, CTB and its receptor the ganglioside, GM1 have been measured in a dilute electrolyte solution, pH 5.5. Although there is variation in the force separation data obtained, particularly on approach of the AFM tip to the GM1 surface where usually, but not always an attraction is noted, an adhesion is always noted on separation of the surfaces. The strength of this adhesion varies from experiment to experiment, but appears to be quantised at a value of around 90 pN. Addition of cholera toxin to the aqueous electrolyte solution completely removes the attractive interaction and adhesion. This gives us confidence that in the earlier experiments, a specific interaction between the CTB and GM1 was measured.