Evaluation of temperature effect on the interaction between β-lactoglobulin and anti-β-lactoglobulin antibody by atomic force microscopy

Molecular recognition such as antigen-antibody interaction is characterized by the parameters of kinetics and the energy landscape. Examinations of molecules involved in the interaction at different temperatures using atomic force microscopy (AFM) can provide information on not only the effects of temperature on the unbinding force between a molecule of interest and a complementary molecule but also the parameters of kinetics and the energy landscape for dissociation of the molecular complex. We investigated the effect of temperature on the dissociation process of the complex of β-lactoglobulin and anti-bovine β-lactoglobulin IgG polyclonal antibody using AFM. Measurements of the unbinding forces between β-lactoglobulin and the antibody were performed at 25, 35, and 45 °C. The following results were obtained in our present study: (i) The unbinding forces decreased as temperature increased, suggesting that the binding force between β-lactoglobulin and the antibody includes the force originating from temperature-dependent interactions (e.g., hydrogen bonding). (ii) At each temperature, the unbinding force exhibited two linear regimes in the force spectra, indicating that the dissociation process of the β-lactoglobulin-antibody complex passes at least two energy barriers from the bound state to the dissociated state. (iii) The dissociation rates at zero force and the position of energy barriers increased as temperature increased. (iv) The heights of the two energy barriers in the reaction coordinates were 49.7 k(B)T and 14.5 k(B)T. (v) The values of roughness of the barriers were ca. 6.1 k(B)T and 3.2 k(B)T. Overall, the present study using AFM revealed more information about the β-lactoglobulin-antibody interaction than studies using conventional bulk measurement such as surface plasmon resonance.     

Can non-mechanical proteins withstand force? Stretching barnase by atomic force microscopy and molecular dynamics simulation

Atomic force microscopy (AFM) experiments have provided intriguing insights into the mechanical unfolding of proteins such as titin I27 from muscle, but will the same be possible for proteins that are not physiologically required to resist force? We report the results of AFM experiments on the forced unfolding of barnase in a chimeric construct with I27. Both modules are independently folded and stable in this construct and have the same thermodynamic and kinetic properties as the isolated proteins. I27 can be identified in the AFM traces based on its previous characterization, and distinct, irregular low-force peaks are observed for barnase. Molecular dynamics simulations of barnase unfolding also show that it unfolds at lower forces than proteins with mechanical function. The unfolding pathway involves the unraveling of the protein from the termini, with much more native-like secondary and tertiary structure being retained in the transition state than is observed in simulations of thermal unfolding or experimentally, using chemical denaturant. Our results suggest that proteins that are not selected for tensile strength may not resist force in the same way as those that are, and that proteins with similar unfolding rates in solution need not have comparable unfolding properties under force.     

Adapting the Quesant© Nomad™ atomic force microscope for biology and patch-clamp atomic force microscopy

The Quesant^© Nomad^? atomic force microscope (AFM) was modified to produce a reliable patch-clamp AFM for demanding biologic applications. The AFM's laser optics forms the basis of a condenser that allows simultaneous Köhler illumination and AFM imaging on an inverted optical microscope. The original AFM scan head was replaced with plastic and glass to make it biologically inert. A bevel cut in the new scan head permits clearance for patch clamp pipets. Cantilevers are attached to the scan head with a quick setting silicone rubber that is readily removable. Software was developed to (a) automate a gentle approach and set a specific feedback force, (b) provide a mouse-driven control of the X-Y position of the probe tip and recall of saved locations, and (c) measure force-distance curves over user defined paths. Additional modifications were made to minimize mechanical noise. The patch-clamp AFM achieves 600 fA (3 kHz bandwidth) and 1 Å RMS noise levels (10 kHz bandwidth). The correlation of electrical and mechanical information allows signal averaging and measures sub-Angstrom, sub-millisecond electromotile responses from cells.     

Submolecular-scale imaging of α-helices and C-terminal domains of tubulins by frequency modulation atomic force microscopy in liquid

In this study, we directly imaged subnanometer-scale structures of tubulins by performing frequency modulation atomic force microscopy (FM-AFM) in liquid. Individual α-helices at the surface of a tubulin protofilament were imaged as periodic corrugations with a spacing of 0.53 nm, which corresponds to the common pitch of an α-helix backbone (0.54 nm). The identification of individual α-helices allowed us to determine the orientation of the deposited tubulin protofilament. As a result, C-terminal domains of tubulins were identified as protrusions with a height of 0.4 nm from the surface of the tubulin. The imaging mechanism for the observed subnanometer-scale contrasts is discussed in relation to the possible structures of the C-terminal domains. Because the C-terminal domains are chemically modified to regulate the interactions between tubulins and other biomolecules (e.g., motor proteins and microtubule-associated proteins), detailed structural information on individual C-terminal domains is valuable for understanding such regulation mechanisms. The results obtained in this study demonstrate that FM-AFM is capable of visualizing the structural variation of tubulins with subnanometer resolution. This is an important first step toward using FM-AFM to analyze the functions of tubulins.     

Dynamic analysis of hepatoma spheroid formation: roles of E-cadherin and β1-integrin

A spheroid is an in vitro multicellular aggregate that provides a microenvironment resembling that of normal tissue in vivo. Although cell adhesion molecules such as integrins and cadherins have been implicated in participating in the process of spheroid formation, little is known about the timing of their action. In this study, we have employed an image-based quantitative method to investigate the compactness of cell aggregates during hepatoma spheroid formation in a dynamic fashion. By modulating β1-integrin and E-cadherin activity with specific blocking antibodies, ion chelators, and RGD-sequence-containing peptides, we show that these cell adhesion molecules mediate the formation of spheroids through the establishment of complex cell-cell and cell-extracellular matrix (ECM) interactions. The dynamics of spheroid formation can be separated into three stages. In the first stage, ECM fibers act as a long-chain linker for the attachment of dispersed single-cells to form loose aggregations through the binding of integrins. This is followed by a delay period in which cell aggregates pause in compaction, presumably because of the accumulation of sufficient amounts of E-cadherins. In the third stage, strong homophilic interaction of E-cadherins is a major factor for the morphological transition from loose cell aggregates to compact spheroids. These findings thus provide comprehensive information on the molecular mechanisms and dynamics of hepatoma spheroid formation.This work was supported by the National Program of Genome Medicine, ROC (NSC 93-3112-B007-001) and Veteran General Hospitals University System of Taiwan Joint Research Program (VGHUST94-G6-06-3).     

Single-molecule atomic force microscopy force spectroscopy study of Aβ-40 interactions

Misfolding and aggregation of amyloid β-40 (Aβ-40) peptide play key roles in the development of Alzheimer's disease (AD). However, very little is known about the molecular mechanisms underlying these molecular processes. We developed a novel experimental approach that can directly probe aggregation-prone states of proteins and their interactions. In this approach, the proteins are anchored to the surface of the atomic force microscopy substrate (mica) and the probe, and the interaction between anchored molecules is measured in the approach-retraction cycles. We used dynamic force spectroscopy (DFS) to measure the stability of transiently formed dimers. One of the major findings from DFS analysis of α-synuclein (α-Syn) is that dimeric complexes formed by misfolded α-Syn protein are very stable and dissociate over a range of seconds. This differs markedly from the dynamics of monomers, which occurs on a microsecond to nanosecond time scale. Here we applied the same approach to quantitatively characterize interactions of Aβ-40 peptides over a broad range of pH values. These studies showed that misfolded dimers are characterized by lifetimes in the range of seconds. This value depends on pH and varies between 2.7 s for pH 2.7 and 0.1 s for pH 7, indicating that the aggregation properties of Aβ-40 are modulated by the environmental conditions. The analysis of the contour lengths revealed the existence of various pathways for dimer dissociation, suggesting that dimers with different conformations are formed. These structural variations result in different aggregation pathways, leading to different types of oligomers and higher-order aggregates, including fibrils.     

Visualization of chromatin folding patterns in chicken erythrocytes by atomic force microscopy （AFM）

The organization of the higher order structure of chromatin in chicken erythrocytes has been examined with tapping-mode scanning force microscopy under conditions close to their native envirinment.Reproducible highresolution AFM images of chromatin compaction at several levels can be demonstrated.An extended beads-on-astring (width of - 15-20nm,height of - 2-3nm for each individual nucleosome) can be consistently observed.Furthermore,superbeade (width of - 40nm,height of - 7nm) are demonstrated.Visualization of the solenoid conformation at the level of 30nm chromatin fiber is attained either by using AFM or by using electron microscopy.In addition,tightly coiled chromatin fibers (- 50-60nm and - 90-110nm) can be revealed.Our data suggest that the chromatin in the interphase nucleus of chicken erythrocyte represents a high-order conformation and AFM provides useful high-resolution structural information concerning the folding pattern of interphase chromatin fibers.     

Effects of point substitutions on the structure of toxic Alzheimer's β-amyloid channels: atomic force microscopy and molecular dynamics simulations

Alzheimer's disease (AD) is a misfolded protein disease characterized by the accumulation of β-amyloid (Aβ) peptide as senile plaques, progressive neurodegeneration, and memory loss. Recent evidence suggests that AD pathology is linked to the destabilization of cellular ionic homeostasis mediated by toxic pores made of Aβ peptides. Understanding the exact nature by which these pores conduct electrical and molecular signals could aid in identifying potential therapeutic targets for the prevention and treatment of AD. Here using atomic force microscopy (AFM) and molecular dynamics (MD) simulations, we compared the imaged pore structures with models to predict channel conformations as a function of amino acid sequence. Site-specific amino acid (AA) substitutions in the wild-type Aβ(1-42) peptide yield information regarding the location and significance of individual AA residues to its characteristic structure-activity relationship. We selected two AAs that our MD simulation predicted to inhibit or permit pore conductance. The substitution of Phe19 with Pro has previously been shown to eliminate conductance in the planar lipid bilayer system. Our MD simulations predict a channel-like shape with a collapsed pore, which is supported by the AFM channel images. We suggest that proline, a known β-sheet breaker, creates a kink in the center of the pore and prevents conductance via blockage. This residue may be a viable target for drug development studies aiming to inhibit Aβ from inducing ionic destabilization toxicity. The substitution of Phe20 with Cys exhibits pore structures indistinguishable from the wild type in AFM images. MD simulations predict site 20 to face the solvated pore. Overall, the mutations support the previously predicted β-sheet-based channel structure.     

In vivo analysis of local wall stiffness at the shoot apical meristem in Arabidopsis using atomic force microscopy

Whereas the morphogenesis of developing organisms is relatively well understood at the molecular level, the contribution of the mechanical properties of the cells to shape changes remains largely unknown, mainly because of the lack of quantified biophysical parameters at cellular or subcellular resolution. Here we designed an atomic force microscopy approach to investigate the elastic modulus of the outer cell wall in living shoot apical meristems (SAMs). SAMs are highly organized structures that contain the plant stem cells, and generate all of the aerial organs of the plant. Building on modeling and experimental data, we designed a protocol that is able to measure very local properties, i.e. within 40-100 nm deep into the wall of living meristematic cells. We identified three levels of complexity at the meristem surface, with significant heterogeneity in stiffness at regional, cellular and even subcellular levels. Strikingly, we found that the outer cell wall was much stiffer at the tip of the meristem (5 ± 2 MPa on average), covering the stem cell pool, than on the flanks of the meristem (1.5 ± 0.7 MPa on average). Altogether, these results demonstrate the existence of a multiscale spatialization of the mechanical properties of the meristem surface, in addition to the previously established molecular and cytological zonation of the SAM, correlating with regional growth rate distribution.     

Organisation of nucleosomal arrays reconstituted with repetitive African green monkey α-satellite DNA as analysed by atomic force microscopy

Alpha-satellite DNA (AS) is part of centromeric DNA and could be relevant for centromeric chromatin structure: its repetitive character may generate a specifically ordered nucleosomal arrangement and thereby facilitate kinetochore protein binding and chromatin condensation. Although nucleosomal positioning on some satellite sequences had been shown, including AS from African green monkey (AGM), the sequence-dependent nucleosomal organisation of repetitive AS of this species has so far not been analysed. We therefore studied the positioning of reconstituted nucleosomes on AGM AS tandemly repeated DNA. Enzymatic analysis of nucleosome arrays formed on an AS heptamer as well as the localisation of mononucleosomes on an AS dimer by atomic force microscopy (AFM) showed one major positioning frame, in agreement with earlier results. The occupancy of this site was in the range of 45–50%, in quite good agreement with published in vivo observations. AFM measurements of internucleosomal distances formed on the heptamer indicated that the nucleosomal arrangement is governed by sequence-specific DNA-histone interactions yielding defined internucleosomal distances, which, nevertheless, are not compatible with a uniform phasing of the nucleosomes with the AGM AS repeats.     

Characterization of testudine melanomacrophage linear,membrane extension processes—Cablepodia—By phase and atomic force microscopy

Summary Melanomacrophages (MMs) are a component of an internal, pigmented cell system in liver and splenic tissues of some fishes, anurans, and reptiles. The cells have been found in centers or aggregates in sinusoids and are associated with cells capable of producing a peptide cytokine and immunoglobulins. A unique cell extension process has been observed in turtle MMs placed into cell culture, and this process has been studied by light and atomic force microscopy. These structures, referred to as cablepodia, are uniquely straight, narrow, and unbranching and appear to originate from growth cones opposite lamellipodia. Cablepodia were found to connect with other turtle MMs and fibroblasts forming cell networks. Dividing fibroblasts to which a cablepodium attached ceased cell division. The observations collectively suggest that a principal reason for aggregations of MMs in internal organs of lower vertebrates in their ability to form interconnected networks of cell processes for trapping and processing of particulate matter, cells and infectious organisms and, possibly, for the communication of cell signals and transfer of intracellular materials.     

Down-regulation of β3-integrin inhibits bone metastasis of small cell lung cancer

Bone is one of the most frequent targets of small cell lung cancer (SCLC) metastasis, but the molecular mechanism remains unclear. β3-integrin plays an important role in invasion of various kinds of tumors. Yet, its role in bone-metastasis of SCLC is still unknown. In this study, we first examined the expression of β3-integrin in SBC-5 and SBC-3 cells by real-time PCR, western blot and immunofluorescence. We found that, compared to none bone-metastatic SBC-3 cells, β3-integrin was highly expressed in SBC-5 cells, a specific bone-metastatic SCLC cells line characterized in our previous study. We next constructed β3-integrin siRNA and transfected SBC-5 cell line, and found that β3-integrin siRNA significantly down-regulated the β3-integrin mRNA level and protein expression in SBC-5 cell line. We further found that inhibition of β3-integrin significantly reduced tumor cell proliferation and induced apoptosis. In addition, the β3-integrin down-regulated cells presented significant decrease in cell adhesion, migration and invasion activity. Our results suggest the β3-integrin has an essential effect on tumor cell proliferation and progression, and may be a potential therapeutic target for the prevention of skeletal metastases of lung cancer.     

Sorting nexin 17 prevents lysosomal degradation of β1 integrins by binding to the β1-integrin tail

Integrin functions are controlled by regulating their affinity for ligand, and by the efficient recycling of intact integrins through endosomes. Here we demonstrate that the Kindlin-binding site in the β1-integrin cytoplasmic domain serves as a molecular switch enabling the sequential binding of two FERM-domain-containing proteins in different cellular compartments. When β1 integrins are at the plasma membrane, Kindlins control ligand-binding affinity. However, when they are internalized, Kindlins dissociate from integrins and sorting nexin 17 (SNX17) is recruited to free β1-integrin tails in early endosomes to prevent β1-integrin degradation, leading to their recycling back to the cell surface. Our results identify SNX17 as a β1-integrin-tail-binding protein that interacts with the free Kindlin-binding site in endosomes to stabilize β1 integrins, resulting in their recycling to the cell surface where they can be reused.     

Atomic force microscopy studies of the adhesive properties of DPPC vesicles containing β-carotene

A role of carotenoids as modulators of physical properties of model and biological membranes has been already postulated. However, there is a lack of information on the influence of these pigments on interactions between the lipids which form such membranes. This paper applies atomic force microscopy (AFM) in to study the effects of β-carotene on the adhesion properties of DPPC multilamellar liposomes. This allowed us to gain, for the first time, a direct insight into the interactions between the components in model systems on a molecular level. We observe that the adhesive forces in DPPC multilamellar liposomes containing 1mol% of β-carotene decrease exponentially with increasing temperature, and that at about 37°C they diminish. In the case of pure liposomes the decline in adhesion is of a different nature and the adhesive forces disappear at 34°C. The adhesive forces are about 5 times higher at 31°C in the presence of β-carotene than in its absence. However, measurements using differential scanning calorimetry (DSC) showed a shift of the lamellar-to-undulled-lamellar phase transition toward lower temperatures by about 0.8 ± 0.2°C in a system containing β-carotene. The enthalpy changes (ΔH) of this transition are similar for both systems. For the main transition, gel-to-liquid crystalline, the peak is shifted by about 0.5 ± 0.1°C, and ΔH decreases by about 30% in liposomes treated with β-carotene in comparison to pure liposomes. Our results suggest increased cooperation between liposome components in a system with enriched β-carotene, which cause a change in phase transition temperatures. Moreover, these interactions are very sensitive to temperature.     

A thin-layer model for viscoelastic, stress-relaxation testing of cells using atomic force microscopy: do cell properties reflect metastatic potential?

Atomic force microscopy has rapidly become a valuable tool for quantifying the biophysical properties of single cells. The interpretation of atomic force microscopy-based indentation tests, however, is highly dependent on the use of an appropriate theoretical model of the testing configuration. In this study, a novel, thin-layer viscoelastic model for stress relaxation was developed to quantify the mechanical properties of chondrosarcoma cells in different configurations to examine the hypothesis that viscoelastic properties reflect the metastatic potential and invasiveness of the cell using three well-characterized human chondrosarcoma cell lines (JJ012, FS090, 105KC) that show increasing chondrocytic differentiation and decreasing malignancy, respectively. Single-cell stress relaxation tests were conducted at 2 h and 2 days after plating to determine cell mechanical properties in either spherical or spread morphologies and analyzed using the new theoretical model. At both time points, JJ012 cells had the lowest moduli of the cell lines examined, whereas FS090 typically had the highest. At 2 days, all cells showed an increase in stiffness and a decrease in apparent viscosity compared to the 2-h time point. Fluorescent labeling showed that the F-actin structure in spread cells was significantly different between FS090 cells and JJ012/105KC cells. Taken together with results of previous studies, these findings indicate that cell transformation and tumorigenicity are associated with a decrease in cell modulus and apparent viscosity, suggesting that cell mechanical properties may provide insight into the metastatic potential and invasiveness of a cell.