Nano-Scale Dynamic Recognition Imaging on Vascular Endothelial Cells

Abstract

Combination of high-resolution atomic force microscope 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. Here simultaneous topography and recognition imaging (TREC) was applied to gently fixed microvascular endothelial cells from mouse myocardium (MyEnd) to identify binding sites of vascular endothelial (VE)-cadherin, known to play a crucial role in calcium-dependent, homophilic cell-to-cell adhesion. TREC images were acquired with magnetically oscillating atomic-force microscope tips functionalized with a recombinant VE-cadherin-Fc cis-dimer. The recognition images revealed single molecular binding sites and prominent, irregularly shaped dark spots (domains) with sizes ranging from 10 to 100 nm. These domains arose from a decrease of the oscillation amplitude during specific binding between active VE-cadherin cis-dimers. The VE-cadherin clusters were subsequently assigned to topography features. TREC represents an exquisite method to quickly obtain the local distribution of receptors on cellular surface with an unprecedented lateral resolution of 5 nm.

Localization of the ergtoxin-1 receptors on the voltage sensing domain of hERG K+ channel by AFM recognition imaging

“…For AFM studies, cells were seeded onto gelatincoated glass slides, grown until 40-50% confluence. To perform TREC measurements, hERG cells were gently fixed with monomeric solution of glutaraldehyde [40].…”

“…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. With the recent development of simultaneous topography and recognition imaging (TREC) [37][38][39] it becomes possible to quickly obtain the local distribution of receptors on cell surface with unprecedented lateral resolution of 5 nm [40]. At present, no other microscopic techniques are able to provide directly both structural information of a biological sample and related functional information at such high spatial resolution.…”

Recent progress in AFM molecular recognition studies

“…This is much greater than the timescale at which dynamic processes usually occur in biology. Fortunately, this problem can be solved by using dynamic recognition imaging [38,39] in which molecular recognition signals are detected during dynamic force microscopy imaging. Topography and recognition (abbreviated as TREC) images are acquired using tips which are magnetically oscillated during scanning and contain ligand molecules directed against the cognate receptor on the surface.…”

“…5b), both recorded at experimental times comparable to normal AFM imaging. Figure 6 shows the application of the simultaneous imaging of topography and recognition sites on cells [39]. Real-time visualization and quantification of receptor binding sites on cell surfaces is a fundamental challenging task in cell biology.…”

“…This is normally achieved by using immunofluorescence techniques such as immunostaining. Recently, the simultaneous imaging of topography and recognition sites has been applied to VE cells to localize VE-cadherin binding sites on the cell membrane [39]. In this study, topography (Fig.…”