Dynamics of podosome stiffness revealed by atomic force microscopy

Podosomes are unique cellular entities specifically found in macrophages and involved in cell-matrix interactions, matrix degradation, and 3D migration. They correspond to a core of F-actin surrounded at its base by matrix receptors. To investigate the structure/function relationships of podosomes, soft lithography, atomic force microscopy (AFM), and correlative fluorescence microscopy were used to characterize podosome physical properties in macrophages differentiated from human blood monocytes. Podosome formation was restricted to delineated areas with micropatterned fibrinogen to facilitate AFM analyses. Podosome height and stiffness were measured with great accuracy in living macrophages (578 ± 209 nm and 43.8 ± 9.3 kPa) and these physical properties were independent of the nature of the underlying matrix. In addition, time-lapse AFM revealed that podosomes harbor two types of overlapping periodic stiffness variations throughout their lifespan, which depend on F-actin and myosin II activity. This report shows that podosome biophysical properties are amenable to AFM, allowing the study of podosomes in living macrophages at nanoscale resolution and the analysis of their intimate dynamics. Such an approach opens up perspectives to better understand the mechanical functionality of podosomes under physiological and pathological contexts.     

Molecular determinants of bacterial adhesion monitored by atomic force microscopy

Bacterial adhesion and the subsequent formation of biofilm are major concerns in biotechnology and medicine. The initial step in bacterial adhesion is the interaction of cells with a surface, a process governed by long-range forces, primarily van der Waals and electrostatic interactions. The precise manner in which the force of interaction is affected by cell surface components and by the physiochemical properties of materials is not well understood. Here, we show that atomic force microscopy can be used to analyze the initial events in bacterial adhesion with unprecedented resolution. Interactions between the cantilever tip and confluent monolayers of isogenic strains of Escherichia coli mutants exhibiting subtle differences in cell surface composition were measured. It was shown that the adhesion force is affected by the length of core lipopolysaccharide molecules on the E. coli cell surface and by the production of the capsular polysaccharide, colanic acid. Furthermore, by modifying the atomic force microscope tip we developed a method for determining whether bacteria are attracted or repelled by virtually any biomaterial of interest. This information will be critical for the design of materials that are resistant to bacterial adhesion.     

Immune synapse formation determines interaction forces between T cells and antigen-presenting cells measured by atomic force microscopy

During adaptive immune responses, T lymphocytes recognize antigenic peptides presented by MHC molecules on antigen-presenting cells (APCs). This recognition results in the formation of a so-called immune synapse (IS) at the T-cell/APC interface, which is crucial for T-cell activation. The molecular composition of the IS has been extensively studied, but little is known about the biophysics and interaction forces between T cells and APCs. Here, we report the measurement of interaction forces between T cells and APCs employing atomic force microscopy (AFM). For these investigations, specific T cells were selected that recognize an antigenic peptide presented by MHC-class II molecules on APCs. Dynamic analysis of T-cell/APC interaction by AFM revealed that in the presence of antigen interaction forces increased from 1 to 2 nN at early time-points to a maximum of ≈14 nN after 30 min and decreased again after 60 min. These data correlate with the kinetics of synapse formation that also reached a maximum after 30 min, as determined by high-throughput multispectral imaging flow cytometry. Because the integrin lymphocyte function antigen-1 (LFA-1) and its counterpart intercellular adhesion molecule-1 (ICAM-1) are prominent members of a mature IS, the effect of a small molecular inhibitor for LFA-1, BIRT377, was investigated. BIRT377 almost completely abolish the interaction forces, emphasizing the importance of LFA-1/ICAM-1-interactions for firm T-cell/APC adhesion. In conclusion, using biophysical measurements, this study provides precise values for the interaction forces between T cells and APCs and demonstrates that these forces develop over time and are highest when synapse formation is maximal.Cell-cell contacts play a crucial role in triggering the body''s immune system. During adaptive immune responses, antigen-presenting cells (APCs) process foreign antigens into peptides, which are loaded into major histocompatibility complex (MHC) molecules. T cells patrolling the body scan APC and establish intercellular contacts when their antigen-specific T-cell receptors (TCR) recognize a foreign peptide/MHC complex on the APC. Elegant two-photon microscopy studies have revealed the dynamics of this process in lymph nodes. There, T cells move through the network of dendritic cells (DCs) and scan DCs for foreign antigen. In the absence of antigen brief transient interactions are observed, whereas upon recognition of a cognate antigen T cells are arrested and interactions prolonged to >1 h (1, 2). Similarly, during antibody responses, long-lasting antigen driven interactions between T helper cells and B cells have been observed in lymph nodes (3). Subsequently, at the contact zone between T cells and APC spatially organized molecular clusters develop, referred to as immune synapse (IS), which is crucial for T-cell activation and effector cells development (4).Formation of an IS includes the coordinated translocation of several protein complexes, among others TCR and its ligand pMHC, and the integrin lymphocyte function-associated antigen-1 (LFA-1) and its counterpart intercellular adhesion molecule 1 (ICAM-1). This orchestrated reorganization of membrane proteins involves many cytoplasmic molecules and is presumably supported by cytoskeletal factors like actin (5). Although many important aspects of IS formation have been identified, little is known about the underlying biophysics and interaction forces between T cells and APCs. Integrins represent a family of major cell adhesion proteins used by cells to tune their adhesion propensity. This tuning is achieved by controlling the number of proteins present at the cell''s interaction face and by the activation state of the adhesion proteins themselves. Switch blade-type heterodimeric integrins are known to exist in different activation states, which are transmitted from the cytoplasmic tail to the extracellular domain (6). It is believed that activation state changes are triggered by inside-out-signaling, for instance when a TCR recognizes a peptide presented by MHC molecules (7). The activation of LFA-1 upon TCR-triggering is mainly mediated by PKC and the small GTPases Ras and Rap1 [(8) and references therein]. The association of actin to LFA-1 accompanies this process. Subsequent motor protein motion yields a cytoskeleton contraction, which exerts low forces on LFA-1 to induce occupied integrin activation and to fully arrest the two cells for adhesion. By actio et reactio, this force has to be counterbalanced on the APC side, resulting in a high interaction force between T cells and APCs.Cell–cell adhesion has been studied by micropipette aspiration techniques (9, 10) and atomic force microscopy (AFM) (11–13). The recent years have seen a significant increase of AFM-related studies in biological systems, and single-cell force spectroscopy (SCFS) by AFM has been established as an important tool for the study of cell adhesion (14). This technique allows for the analysis of adhesion processes and adhesion forces under near-physiological conditions. To the best of our knowledge, the interaction forces between T cells and APC have not yet been investigated by SCFS. In the present study, we have modified and adjusted SCFS techniques for the measurement of long-time interaction forces between T cells and APC. These force spectroscopy measurements were complemented by conjugate and high-throughput fluorescence assays relating the kinetics of IS formation to the development of interaction forces between T cells and APCs.     

原子力显微镜在双微体研究中的方法学探讨

目的探讨影响原子力显微镜(atomic force microscopy，AFM)观察双微体(double minute chromosomes，DMs)的各种因素，包括原子力显微镜成像模式的选择，以及样品制备过程中各种条件的优化。方法培养耐氨甲喋呤(MTX)300μmol／L的小鼠成纤维细胞系313R500，收获中期染色体后制片，胃蛋白酶消化标本，梯度酒精脱水，最后用原子力显微镜观察染色体中期分裂相。结果优化条件后，在原子力显微镜下很容易找到分裂相。图像扫描显示，染色体在分散程度和清晰度上都有很大提高，双微体的成像质量也有极大的改善。结论优化染色体制片条件及选择恰当的原子力显微镜成像模式可获得高质量的DMs图片。     

Interactions between synaptic vesicle fusion proteins explored by atomic force microscopy

Measuring the biophysical properties of macromolecular complexes at work is a major challenge of modern biology. The protein complex composed of vesicle-associated membrane protein 2, synaptosomal-associated protein of 25 kDa, and syntaxin 1 [soluble N-ethyl-maleimide-sensitive factor attachment protein receptor (SNARE) complex] is essential for docking and fusion of neurotransmitter-filled synaptic vesicles with the presynaptic membrane. To better understand the fusion mechanisms, we reconstituted the synaptic SNARE complex in the imaging chamber of an atomic force microscope and measured the interaction forces between its components. Each protein was tested against the two others, taken either individually or as binary complexes. This approach allowed us to determine specific interaction forces and dissociation kinetics of the SNAREs and led us to propose a sequence of interactions. A theoretical model based on our measurements suggests that a minimum of four complexes is probably necessary for fusion to occur. We also showed that the regulatory protein neuronal Sec1 injected into the atomic force microscope chamber prevented the complex formation. Finally, we measured the effect of tetanus toxin protease on the SNARE complex and its activity by on-line registration during tetanus toxin injection. These experiments provide a basis for the functional study of protein microdomains and also suggest opportunities for sensitive screening of drugs that can modulate protein-protein interactions.     

Revealing the hidden atom in graphite by low-temperature atomic force microscopy

Carbon, the backbone material of life on Earth, comes in three modifications: diamond, graphite, and fullerenes. Diamond develops tetrahedral sp3 bonds, forming a cubic crystal structure, whereas graphite and fullerenes are characterized by planar sp2 bonds. Polycrystalline graphite is the basis for many products of everyday life: pencils, lubricants, batteries, arc lamps, and brushes for electric motors. In crystalline form, highly oriented pyrolytic graphite is used as a diffracting element in monochromators for x-ray and neutron scattering and as a calibration standard for scanning tunneling microscopy (STM). The graphite surface is easily prepared as a clean atomically flat surface by cleavage. This feature is attractive and is used in many laboratories as the surface of choice for "seeing atoms." Despite the proverbial ease of imaging graphite by STM with atomic resolution, every second atom in the hexagonal surface unit cell remains hidden, and STM images show only a single atom in the unit cell. Here we present measurements with a low-temperature atomic force microscope with pico-Newton force sensitivity that reveal the hidden surface atom.     

Application of atomic force microscopy in blood research

AIM: To find suitable solutions having lesser granules and keeping erythrocytes in normal shapes under atomic force microscopy (AFM). METHODS: Eight kinds of solutions, 1% formaldehyde, PBS buffer (pH7.2), citrate buffer (pH6.0), 0.9% NaCl, 5% dextrose, TAE, 1640 medium and 5% EDTA-K2, were selected from commonly used laboratory solutions, and venous blood from a healthy human volunteer was drawn and anticoagulated with EDTA-K2. Before scanned by AFM (NanoScopeIIIa SPM, Digital Instruments, Santa Barbara, CA), a kind of intermixture was deposited on freshly cleaved mica and then dried in the constant temperature cabinet (37 ℃). RESULTS: One percent formaldehyde, citrate buffer, 5% dextrose, TAE, were found to keep human erythrocytes in normal shape with few particles. Processed by these solutions, fine structures of human erythrocyte membrane were obtained. CONCLUSION: One percent formaldehyde, citrate buffer, 5% dextrose and TAE may be applied to dispose erythrocytes in AFM. The results may offer meaningful data for clinical diagnosis of blood by AFM.     

Monitoring the assembly of Ig light-chain amyloid fibrils by atomic force microscopy

Aggregation of Ig light chains to form amyloid fibrils is a characteristic feature of light-chain amyloidosis, a light-chain deposition disease. A recombinant variable domain of the light chain SMA was used to form amyloid fibrils in vitro. Fibril formation was monitored by atomic force microscopy imaging. Single filaments 2.4 nm in diameter were predominant at early times; protofibrils 4.0 nm in diameter were predominant at intermediate times; type I and type II fibrils 8.0 nm and 6.0 nm in diameter, respectively, were predominant at the endpoints. The increase in number of fibrils correlated with increased binding of the fluorescent dye thioflavin T. The fibrils and protofibrils showed a braided structure, suggesting that their formation involves the winding of protofibrils and filaments, respectively. These observations support a model in which two filaments combine to form a protofibril, two protofibrils intertwine to form a type I fibril, and three filaments form a type II fibril.     

Noncontact estimation of intercellular breaking force using a femtosecond laser impulse quantified by atomic force microscopy

When a femtosecond laser pulse (fsLP) is focused through an objective lens into a culture medium, an impulsive force (fsLP-IF) is generated that propagates from the laser focal point (O_f) in a micron-sized space. This force can detach individual adherent cells without causing considerable cell damage. In this study, an fsLP-IF was reflected in the vibratory movement of an atomic force microscopy (AFM) cantilever. Based on the magnitude of the vibration and the geometrical relationship between O_f and the cantilever, the fsLP-IF generated at O_f was calculated as a unit of impulse [N-s]. This impulsive force broke adhesion molecule-mediated intercellular interactions in a manner that depended on the adhesion strength that was estimated by the cell aggregation assay. The force also broke the interactions between streptavidin-coated microspheres and a biotin-coated substrate with a measurement error of approximately 7%. These results suggest that fsLP-IF can be used to break intermolecular and intercellular interactions and estimate the adhesion strength. The fsLP-IF was used to break intercellular contacts in two biologically relevant cultures: a coculture of leukocytes seeded over on an endothelial cell monolayer, and a polarized monolayer culture of epithelial cells. The impulses needed to break leukocyte–endothelial and interepithelial interactions, which were calculated based on the geometrical relationship between O_f and the adhesive interface, were on the order of 10^-13 and 10^-12 N-s, respectively. When the total impulse at O_f is well-defined, fsLP-IF can be used to estimate the force required to break intercellular adhesions in a noncontact manner under biologically relevant conditions.     

Stepwise unfolding of titin under force-clamp atomic force microscopy

Here we demonstrate the implementation of a single-molecule force clamp adapted for use with an atomic force microscope. We show that under force-clamp conditions, an engineered titin protein elongates in steps because of the unfolding of its modules and that the waiting times to unfold are exponentially distributed. Force-clamp measurements directly measure the force dependence of the unfolding probability and readily captures the different mechanical stability of the I27 and I28 modules of human cardiac titin. Force-clamp spectroscopy promises to be a direct way to probe the mechanical stability of elastic proteins such as those found in muscle, the extracellular matrix, and cell adhesion.     

Detection and localization of individual antibody-antigen recognition events by atomic force microscopy.

A methodology has been developed for the study of molecular recognition at the level of single events and for the localization of sites on biosurfaces, in combining force microscopy with molecular recognition by specific ligands. For this goal, a sensor was designed by covalently linking an antibody (anti-human serum albumin, polyclonal) via a flexible spacer to the tip of a force microscope. This sensor permitted detection of single antibody-antigen recognition events by force signals of unique shape with an unbinding force of 244 +/- 22 pN. Analysis revealed that observed unbinding forces originate from the dissociation of individual Fab fragments from a human serum albumin molecule. The two Fab fragments of the antibody were found to bind independently and with equal probability. The flexible linkage provided the antibody with a 6-nm dynamical reach for binding, rendering binding probability high, 0.5 for encounter times of 60 ms. This permitted fast and reliable detection of antigenic sites during lateral scans with a positional accuracy of 1.5 nm. It is indicated that this methodology has promise for characterizing rate constants and kinetics of molecular recognition complexes and for molecular mapping of biosurfaces such as membranes.     

Energy levels of few-electron quantum dots imaged and characterized by atomic force microscopy

Strong confinement of charges in few-electron systems such as in atoms, molecules, and quantum dots leads to a spectrum of discrete energy levels often shared by several degenerate states. Because the electronic structure is key to understanding their chemical properties, methods that probe these energy levels in situ are important. We show how electrostatic force detection using atomic force microscopy reveals the electronic structure of individual and coupled self-assembled quantum dots. An electron addition spectrum results from a change in cantilever resonance frequency and dissipation when an electron tunnels on/off a dot. The spectra show clear level degeneracies in isolated quantum dots, supported by the quantitative measurement of predicted temperature-dependent shifts of Coulomb blockade peaks. Scanning the surface shows that several quantum dots may reside on what topographically appears to be just one. Relative coupling strengths can be estimated from these images of grouped coupled dots.     

Segmented nanofibers of spider dragline silk: atomic force microscopy and single-molecule force spectroscopy

Despite its remarkable materials properties, the structure of spider dragline silk has remained unsolved. Results from two probe microscopy techniques provide new insights into the structure of spider dragline silk. A soluble synthetic protein from dragline silk spontaneously forms nanofibers, as observed by atomic force microscopy. These nanofibers have a segmented substructure. The segment length and amino acid sequence are consistent with a slab-like shape for individual silk protein molecules. The height and width of nanofiber segments suggest a stacking pattern of slab-like molecules in each nanofiber segment. This stacking pattern produces nano-crystals in an amorphous matrix, as observed previously by NMR and x-ray diffraction of spider dragline silk. The possible importance of nanofiber formation to native silk production is discussed. Force spectra for single molecules of the silk protein demonstrate that this protein unfolds through a number of rupture events, indicating a modular substructure within single silk protein molecules. A minimal unfolding module size is estimated to be around 14 nm, which corresponds to the extended length of a single repeated module, 38 amino acids long. The structure of this spider silk protein is distinctly different from the structures of other proteins that have been analyzed by single-molecule force spectroscopy, and the force spectra show correspondingly novel features.     

液相中炭疽杆菌形貌的原子力显微镜观测

目的利用原子力显微镜液相模式观测炭疽杆菌形态,获得液相中炭疽杆菌芽胞和繁殖体对于溶菌酶的形态学抗性变化数据。方法应用原子力显微镜液相模式观察炭疽杆菌繁殖体及芽胞的超微结构,并对其主要指标进行测量比较。结果1×PBS溶液中的炭疽杆菌繁殖体,菌体杆状,竹节样排列,菌体间连接致密,长度为2,757.30±1227.086nm,宽度为1,710.90±226.10nm,Rq为15.447±3.418nm,Ra为13.239±2.733nm;炭疽杆菌芽胞椭圆形,多散在分布,表面平滑,长度为2,014.00±227.155nm,宽度为1,264.10±132.180nm,Rq为22.803±5.660nm,Ra为18.010±4.568nm。0.01 mg/mL溶菌酶溶液中的炭疽杆菌繁殖体形态不规则,表面凹凸起伏,边缘粗糙,有囊状突起,长度为2,836.00±1025.137nm,宽度为2,449.00±212.78nm,Rq为17.068±4.427nm,Ra为14.776±3.746nm;0.01 mg/mL溶菌酶溶液中的炭疽杆菌芽胞,形态规则,边缘平滑,未见凹凸起伏和囊状突起,而1 mg/mL溶菌酶溶液环境中的炭疽杆菌芽胞形态不规则,表面起伏,变化较大,其长度为2,155.00±202.663nm,宽度为1,344.00±162.631nm,Rq为24.849±3.427nm,Ra为20.869±2.550nm。结论通过原子力显微镜液相模式下的观察和测量,清楚地显示了溶液中炭疽杆菌的微观形貌及其变化,0.01 mg/mL的溶菌酶就能使炭疽杆菌繁殖体发生形态学变化,而炭疽杆菌芽胞形貌发生变化则需要1 mg/mL的高浓度溶菌酶的作用。     

Continuous detection of extracellular ATP on living cells by using atomic force microscopy.

Atomic force microscopy is a powerful technique used to investigate the surface of living cells under physiological conditions. The resolution of the instrument is mainly limited by the softness of living cells and the interactions with the scanning tip (cantilever). Atomic force microscopy, in combination with myosin-functionalized cantilevers, was used in the detection of ATP concentrations in solution and on living cells. Functionally active tips were used to scan the surface of cells in culture and to show that the CFTR+ cell line (S9) had a basal surface ATP concentration that could be detected with atomic force microscopy (n = 10). ATP-dependent signals were not detectable in cells scanned with noncoated or heat-inactivated enzyme-coated tips (n = 9). Enzymatically active tips may serve as a model for future development of atomic force microscopy biosensors that can simultaneously detect topographical and biologically important compounds at the surface microenvironment of living cells.     

Direct measurement of hydrogen bonding in DNA nucleotide bases by atomic force microscopy.

We have used self-assembled purines and pyrimidines on planar gold surfaces and on gold-coated atomic force microscope (AFM) tips to directly probe intermolecular hydrogen bonds. Electron spectroscopy for chemical analysis (ESCA) and thermal programmed desorption (TPD) measurements of the molecular layers suggested monolayer coverage and a desorption energy of about 25 kcal/mol. Experiments were performed under water, with all four DNA bases immobilized on AFM tips and flat surfaces. Directional hydrogen-bonding interaction between the tip molecules and the surface molecules could be measured only when opposite base-pair coatings were used. The directional interactions were inhibited by excess nucleotide base in solution. Nondirectional van der Waals forces were present in all other cases. Forces as low as two interacting base pairs have been measured. With coated AFM tips, surface chemistry-sensitive recognition atomic force microscopy can be performed.     

Unbinding process of adsorbed proteins under external stress studied by atomic force microscopy spectroscopy

We report the study of the dynamics of the unbinding process under a force load f of adsorbed proteins (fibrinogen) on a solid surface (hydrophilic silica) by means of atomic force microscopy spectroscopy. By varying the loading rate r(f), defined by f = r(f) t, t being the time, we find that, as for specific interactions, the mean rupture force increases with r(f). This unbinding process is analyzed in the framework of the widely used Bell model. The typical dissociation rate at zero force entering in the model lies between 0. 02 and 0.6 s(-1). Each measured rupture is characterized by a force f(0), which appears to be quantized in integer multiples of 180-200 pN.