Lilia A. Chtcheglova

Force spectroscopy with a small dithering of AFM tip: A method of direct and continuous measurement of the spring constant of single molecules and molecular complexes

A new method of direct and continuous measurement of the spring constant of single molecule or molecular complex is elaborated. To that end the standard force spectroscopy technique with functionalized tips and samples is combined with a small dithering of the tip. The change of the dithering amplitude as a function of the pulling force is measured to extract the spring constant of the complex. The potentialities of this method are illustrated for the experiments with single bovine serum albumin-its polyclonal antibody (Ab-BSA) and fibrinogen-fibrinogen complexes.

Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging

The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on alpha-galactosylceramide (alphaGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from approximately 25 to approximately 160 nm, with the smaller domains corresponding to a single CD1d molecule.

Simultaneous topography and recognition imaging: physical aspects and optimal imaging conditions

Simultaneous topography and recognition imaging (TREC) allows for the investigation of receptor distributions on natural biological surfaces under physiological conditions. Based on atomic force microscopy (AFM) in combination with a cantilever tip carrying a ligand molecule, it enables us to sense topography and recognition of receptor molecules simultaneously with nanometre accuracy. In this study we introduce optimized handling conditions and investigate the physical properties of the cantilever-tip-sample ensemble, which is essential for the interpretation of the experimental data gained from this technique. In contrast to conventional AFM methods, TREC is based on a more sophisticated feedback loop, which enables us to discriminate topographical contributions from recognition events in the AFM cantilever motion. The features of this feedback loop were investigated through a detailed analysis of the topography and recognition data obtained on a model protein system. Single avidin molecules immobilized on a mica substrate were imaged with an AFM tip functionalized with a biotinylated IgG. A simple procedure for adjusting the optimal amplitude for TREC imaging is described by exploiting the sharp localization of the TREC signal within a small range of oscillation amplitudes. This procedure can also be used for proving the specificity of the detected receptor-ligand interactions. For understanding and eliminating topographical crosstalk in the recognition images we developed a simple theoretical model, which nicely explains its origin and its dependence on the excitation frequency.

AFM functional imaging on vascular endothelial cells

Vascular endothelial (VE)‐cadherin is predominantly responsible for the mechanical linkage between endothelial cells, where VE‐cadherin molecules are clustered and linked through their cytoplasmic domain to the actin‐based cytoskeleton. Clustering and linkage of VE‐cadherin to actin filaments is a dynamic process and changes according to the functional state of the cells. Here nano‐mapping of VE‐cadherin was performed using simultaneous topography and recognition imaging (TREC) technique onto microvascular endothelial cells from mouse myocardium (MyEnd). The recognition maps revealed prominent ‘dark’ spots (domains or clusters) with the sizes from 10 to 250 nm. These spots arose from a decrease of oscillation amplitude during specific binding between VE‐cadherin cis‐dimers. They were assigned to characteristic structures of the topography images. After treatment with nocodazole so as to depolymerize microtubules, VE‐cadherin domains with a typical ellipsoidal form were still found to be collocalized with cytoskeletal filaments supporting the hypothesis that VE‐cadherin is linked to actin filaments. Compared to other conventional techniques such as immunochemistry or single molecule optical microscopy, TREC represents an alternative method to quickly obtain the local distribution of receptors on cell surface with an unprecedented lateral resolution of several nanometers. Copyright

Nanoscale Organization of Human GnRH-R on Human Bladder Cancer Cells

Human gonadotropin-releasing hormone receptor (GnRH-R; or type I GnRH-R), which is expressed in tumor cells, has gained more and more attention as a specific target for cancer therapy. Given the clinical utility, the improved characterization of both the subcellular distribution and surface organization of GnRH-R is an important step in the development of more effective and possibly new therapeutic strategies. In the present study, the nano-organization of human GnRH-R was analyzed on fixed human bladder cancer cells (T24) by atomic force microscopy (AFM). The recognition images reveal that GnRH-Rs have a tendency to assemble in nanodomains (or clusters) that are irregularly distributed on the T24 cell surface. The locations of the GnRH-Rs were identified on the topographical images with nanometer accuracy. The obtained results enrich our understanding of the local distribution of GnRH-Rs on the bladder cancer cell membrane and demonstrate the ability of biological AFM to provide more complete and exact information at the single molecule level.

Molecular recognition imaging using tuning fork-based transverse dynamic force microscopy.

We demonstrate simultaneous transverse dynamic force microscopy and molecular recognition imaging using tuning forks as piezoelectric sensors. Tapered aluminum-coated glass fibers were chemically functionalized with biotin and anti-lysozyme molecules and attached to one of the prongs of a 32kHz tuning fork. The lateral oscillation amplitude of the tuning fork was used as feedback signal for topographical imaging of avidin aggregates and lysozyme molecules on mica substrate. The phase difference between the excitation and detection signals of the tuning fork provided molecular recognition between avidin/biotin or lysozyme/anti-lysozyme. Aggregates of avidin and lysozyme molecules appeared as features with heights of 1-4nm in the topographic images, consistent with single molecule atomic force microscopy imaging. Recognition events between avidin/biotin or lysozyme/anti-lysozyme were detected in the phase image at high signal-to-noise ratio with phase shifts of 1-2 degrees. Because tapered glass fibers and shear-force microscopy based on tuning forks are commonly used for near-field scanning optical microscopy (NSOM), these results open the door to the exciting possibility of combining optical, topographic and biochemical recognition at the nanometer scale in a single measurement and in liquid conditions.

Second harmonic atomic force microscopy imaging of live and fixed mammalian cells.

Higher harmonic contributions in the movement of an oscillating atomic force microscopy (AFM) cantilever are generated by nonlinear tip-sample interactions, yielding additional information on structure and physical properties such as sample stiffness. Higher harmonic amplitudes are strongly enhanced in liquid compared to the operation in air, and were previously reported to result in better structural resolution in highly organized lattices of proteins in bacterial S-layers and viral capsids [J. Preiner, J. Tang, V. Pastushenko, P. Hinterdorfer, Phys. Rev. Lett. 99 (2007) 046102]. We compared first and second harmonics AFM imaging of live and fixed human lung epithelial cells, and microvascular endothelial cells from mouse myocardium (MyEnd). Phase-distance cycles revealed that the second harmonic phase is 8 times more sensitive than the first harmonic phase with respect to variations in the distance between cantilever and sample surface. Frequency spectra were acquired at different positions on living and fixed cells with second harmonic amplitude values correlating with the sample stiffness. We conclude that variations in sample stiffness and corresponding changes in the cantilever-sample distance, latter effect caused by the finite feedback response, result in second harmonic images with improved contrast and information that is not attainable in the fundamental frequency of an oscillating cantilever.

Single Molecule Force Microscopy on Cells and Biological Membranes

Atomic force microscopy (AFM) enables high resolution topographic imaging of biological samples under near-physiological conditions. Therefore, the AFM is optimally suited for investigation of biological membranes and cell surfaces, as exemplified by studies on bacterial S-layers, purple membranes and cultured living cells. Topographic imaging allows visualizing single proteins and protein assemblies in native membranes, as well as substructures of live cells, such as cytoskeletal architecture. In addition to high-resolution imaging, the measurement of mechanical forces yields detailed insight into structure-function relationships of molecular processes in their native environment. In molecular recognition force microscopy, interaction forces between tip-bound ligands and membrane-embedded receptors can be studied under well-controlled buffer conditions and effectors concentrations. In case of low lateral density and inhomogeneous distribution of the target molecules in a cell membrane, fluorescence microscopy can help to guide the AFM tip to the membrane proteins of interest, which can subsequently be investigated by molecular recognition force microscopy.

Cell surface localised Hsp70 is a cancer specific regulator of clathrin-independent endocytosis

The stress inducible heat shock protein 70 (Hsp70) is present specifically on the tumour cell surface yet without a pro‐tumour function revealed. We show here that cell surface localised Hsp70 (sHsp70) supports clathrin‐independent endocytosis (CIE) in melanoma models. Remarkably, ability of Hsp70 to cluster on lipid raftsin vitro correlated with larger nano‐domain sizes of sHsp70 in high sHsp70 expressing cell membranes. Interfering with Hsp70 oligomerisation impaired sHsp70‐mediated facilitation of endocytosis. Altogether our findings suggest that a sub‐fraction of sHsp70 co‐localising with lipid rafts enhances CIE through oligomerisation and clustering. Targeting or utilising this tumour specific mechanism may represent an additional benefit for anti‐cancer therapy.

Characterizing the S-layer structure and anti-S-layer antibody recognition on intact Tannerella forsythia cells by scanning probe microscopy and small angle X-ray scattering

Tannerella forsythia is among the most potent triggers of periodontal diseases, and approaches to understand underlying mechanisms are currently intensively pursued. A ~22‐nm‐thick, 2D crystalline surface (S‐) layer that completely covers Tannerella forsythia cells is crucially involved in the bacterium–host cross‐talk. The S‐layer is composed of two intercalating glycoproteins (TfsA‐GP, TfsB‐GP) that are aligned into a periodic lattice. To characterize this unique S‐layer structure at the nanometer scale directly on intact T. forsythia cells, three complementary methods, i.e., small‐angle X‐ray scattering (SAXS), atomic force microscopy (AFM), and single‐molecular force spectroscopy (SMFS), were applied. SAXS served as a difference method using signals from wild‐type and S‐layer‐deficient cells for data evaluation, revealing two possible models for the assembly of the glycoproteins. Direct high‐resolution imaging of the outer surface of T. forsythia wild‐type cells by AFM revealed a p4 structure with a lattice constant of ~9.0 nm. In contrast, on mutant cells, no periodic lattice could be visualized. Additionally, SMFS was used to probe specific interaction forces between an anti‐TfsA antibody coupled to the AFM tip and the S‐layer as present on T. forsythia wild‐type and mutant cells, displaying TfsA‐GP alone. Unbinding forces between the antibody and wild‐type cells were greater than with mutant cells. This indicated that the TfsA‐GP is not so strongly attached to the mutant cell surface when the co‐assembling TfsB‐GP is missing. Altogether, the data gained from SAXS, AFM, and SMFS confirm the current model of the S‐layer architecture with two intercalating S‐layer glycoproteins and TfsA‐GP being mainly outwardly oriented. Copyright