Quasistatic and dynamic force microscopy of single antigen-antibody complexes and fibrin-fibrinogen systems

The molecular processes based on the specific receptor-ligand interactions (e.g. immune response to a foreign antigen, communication between nerve cells, etc.) are essential for a good and stable performance of living organisms. The aim of the present work is to study such molecular processes with the Atomic Force Microscope (AFM) operated in Force Spectroscopy (FS) mode. Information such as the force and length of the ligand-receptor bond rupture could be obtained from the standard quasistatic FS studies, whereas no external dithering applied to the AFM tips. Firstly, the antigen-antibody interactions were examined with the Bovine Serum Albumin (BSA) protein and its poly- and two different monoclonal antibodies. It was shown that the peak unbinding force depends on the type of antibody and the antibody concentration. The single potential barrier is dominant for the interaction between BSA and monoclonal antibodies and was revealed from the loading rate-dependent measurements. Secondly, we are interested in the process of fibrin gel formation, in particular in the forces involved in this process. It was demonstrated that the fibrin-fibrinogen interaction is the specific one. The unbinding force rupture for single fibrin-fibrin(ogen) complex was determined to be of about 320 pN at a loading rate of 3.5 nN/s. The single potential barrier is also dominant for this interaction. Moreover, a new method of direct and continuous measurement of the spring constant of single molecules or molecular complexes has been elaborated in our laboratory. To this end, the standard FS technique with functionalized tips and samples is combined with a small dithering of the AFM tip. The change of the dithering amplitude as a function of the pulling force is measured in order to extract the spring constant of the complex. The potentialities of this technique were demonstrated for the experiments with single BSA – polyclonal antibody to BSA (Ab-BSA) and fibrinogen – fibrinogen complexes

The Interplay between Chemistry and Mechanics in the Transduction of a Mechanical Signal into a Biochemical Function

There are many processes in biology in which mechanical forces are generated. Force-bearing networks can transduce locally developed mechanical signals very extensively over different parts of the cell or tissues. In this article we conduct an overview of this kind of mechanical transduction, focusing in particular on the multiple layers of complexity displayed by the mechanisms that control and trigger the conversion of a mechanical signal into a biochemical function. Single molecule methodologies, through their capability to introduce the force in studies of biological processes in which mechanical stresses are developed, are unveiling subtle intertwining mechanisms between chemistry and mechanics and in particular are revealing how chemistry can control mechanics. The possibility that chemistry interplays with mechanics should be always considered in biochemical studies.Comment: 50 pages, 18 figure

Rickettsiae Induce Microvascular Hyperpermeability via Phosphorylation of VE-Cadherins: Evidence from Atomic Force Microscopy and Biochemical Studies

The most prominent pathophysiological effect of spotted fever group (SFG) rickettsial infection of microvascular endothelial cells (ECs) is an enhanced vascular permeability, promoting vasogenic cerebral edema and non-cardiogenic pulmonary edema, which are responsible for most of the morbidity and mortality in severe cases. To date, the cellular and molecular mechanisms by which SFG Rickettsia increase EC permeability are largely unknown. In the present study we used atomic force microscopy (AFM) to study the interactive forces between vascular endothelial (VE)-cadherin and human cerebral microvascular EC infected with R. montanensis, which is genetically similar to R. rickettsii and R. conorii, and displays a similar ability to invade cells, but is non-pathogenic and can be experimentally manipulated under Biosafety Level 2 (BSL2) conditions. We found that infected ECs show a significant decrease in VE-cadherin-EC interactions. In addition, we applied immunofluorescent staining, immunoprecipitation phosphorylation assay, and an in vitro endothelial permeability assay to study the biochemical mechanisms that may participate in the enhanced vascular permeability as an underlying pathologic alteration of SFG rickettsial infection. A major finding is that infection of R. montanensis significantly activated tyrosine phosphorylation of VE-cadherin beginning at 48 hr and reaching a peak at 72 hr p.i. In vitro permeability assay showed an enhanced microvascular permeability at 72 hr p.i. On the other hand, AFM experiments showed a dramatic reduction in VE-cadherin-EC interactive forces at 48 hr p.i. We conclude that upon infection by SFG rickettsiae, phosphorylation of VE-cadherin directly attenuates homophilic protein–protein interactions at the endothelial adherens junctions, and may lead to endothelial paracellular barrier dysfunction causing microvascular hyperpermeability. These new approaches should prove useful in characterizing the antigenically related SFG rickettsiae R. conorii and R. rickettsii in a BSL3 environment. Future studies may lead to the development of new therapeutic strategies to inhibit the VE-cadherin-associated microvascular hyperpermeability in SFG rickettsioses

Variations on Fibrinogen-Erythrocyte Interactions during Cell Aging

Erythrocyte hyperaggregation, a cardiovascular risk factor, is considered to be caused by an increase in plasma adhesion proteins, particularly fibrinogen. We have recently reported a specific binding between fibrinogen and an erythrocyte integrin receptor with a β3 or β3-like subunit. In this study we evaluate the influence of erythrocyte aging on the fibrinogen binding. By atomic force microscopy-based force spectroscopy measurements we found that increasing erythrocyte age, there is a decrease of the binding to fibrinogen by decreasing the frequency of its occurrence but not its force. This observation is reinforced by zeta-potential and fluorescence spectroscopy measurements. We conclude that upon erythrocyte aging the number of fibrinogen molecules bound to each cell decreases significantly, due to the progressive impairment of the specific fibrinogen-erythrocyte receptor interaction. Knowing that younger erythrocytes bind more to fibrinogen, we could presume that this population is the main contributor to the cardiovascular diseases associated with increased fibrinogen content in blood, which could disturb the blood flow. Our data also show that the sialic acids exposed on the erythrocyte membrane contribute for the interaction with fibrinogen, possibly by facilitating its binding to the erythrocyte membrane receptor

Biophysical Assessment of Single Cell Cytotoxicity: Diesel Exhaust Particle-Treated Human Aortic Endothelial Cells

Exposure to diesel exhaust particles (DEPs), a major source of traffic-related air pollution, has become a serious health concern due to its adverse influences on human health including cardiovascular and respiratory disorders. To elucidate the relationship between biophysical properties (cell topography, cytoskeleton organizations, and cell mechanics) and functions of endothelial cells exposed to DEPs, atomic force microscope (AFM) was applied to analyze the toxic effects of DEPs on a model cell line from human aortic endothelial cells (HAECs). Fluorescence microscopy and flow cytometry were also applied to further explore DEP-induced cytotoxicity in HAECs. Results revealed that DEPs could negatively impair cell viability and alter membrane nanostructures and cytoskeleton components in a dosage- and a time-dependent manner; and analyses suggested that DEPs-induced hyperpolarization in HAECs appeared in a time-dependent manner, implying DEP treatment would lead to vasodilation, which could be supported by down-regulation of cell biophysical properties (e.g., cell elasticity). These findings are consistent with the conclusion that DEP exposure triggers important biochemical and biophysical changes that would negatively impact the pathological development of cardiovascular diseases. For example, DEP intervention would be one cause of vasodilation, which will expand understanding of biophysical aspects associated with DEP cytotoxicity in HAECs

Force Spectroscopy of Polyclonal and Monoclonal Anti-Bovine Serum Albumin Antibodies - BSA Complexes

The specific interactions between bovine serum albumin and poly- or two monoclonal bovine serum albumin antibodies were studied using force spectroscopy mode of atomic force microscopy. The histograms of the unbinding forces for polyclonal bovine serum albumin antibodies are broad at high antibody concentrations (50 or 270 μg/ml) and narrow at low concentrations (10 or 27 μg/ml), while the histograms for monoclonal antibodies peak at well defined unbinding force. The peak unbinding force depends on the type of antibody and the antibody concentration. In this paper we described and characterized the passive adsorption and covalent immobilization of proteins for tip and sample preparation. Force spectroscopy could serve as a useful method for characterization of antigen-antibody interactions for measuring the specificity of an antibody or to assess the purity of a monoclonal antibody solution and to distinguish between different antibodies