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.

FIGURE 1

Scheme of dynamic recognition imaging to visualize VE-cadherin binding sites (here single cis-dimers and/or clusters) on a gently fixed MyEnd cell surface.

FIGURE 2

Recognition images of a MyEnd cell surface obtained with VE-cadherin-Fc-functionalized tip. (A–C) During 1 h of scanning recognition maps of VE-cadherin domains remain unchanged. Then, 5 mM EDTA was very slowly (∼50 μl/min) injected in the fluid cell while scanning the sample. The first scan (D) did not reveal immediate changes in the recognition map. The recognition clusters disappeared as the active VE-cadherin-Fc cis-dimers on the AFM tip dissociated in inactive monomers, thereby abolishing specific VE-cadherin trans-interaction (E–H). Note that in Ca²⁺-rich conditions the previously blocked tip regains its functionality. (B′,F′) Topography images simultaneously recorded with B and F, respectively. After blocking experiments, topography (F′) remains unchanged—indicating that blocking does not affect membrane topography (compare B′ and F′). Red stars in B′ and F′ indicate the AFM scanner lateral drift of ∼5 nm/min. (B+, B++) Examples of recognition spots magnified from B (areas + and ++, respectively). Recognition areas are depicted by threshold analysis (threshold = −1.7 nm) and bordered by white lines. Single VE-cadherin cis-dimers can be clearly detected (arrows).

FIGURE 3

Superimposition of recognition map of VE-cadherin domains (in green) onto the corresponding topography image. Color scale (dark brown to white) is 0–12 nm.

FIGURE 4

Force distribution recorded on gently fixed MyEnd surface with VE-cadherin-Fc-coated tip in Ca²⁺-rich conditions.