AFM functional imaging on vascular endothelial cells

Abstract

a 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.

N-Glycosylation Alters Cadherin-Mediated Intercellular Binding Kinetics

“…At cell-cell junctions, cadherins form clusters involving many hundreds of proteins (Chtcheglova et al, 2010;Shewan et al, 2005;Yap et al, 1997a;Yap et al, 1997b). However, clustering requires active processes, and is unlikely driven by protein-protein interactions (Shewan et al, 2005).…”

Molecular Recognition Force Spectroscopy Study of the Specific Lectin and Carbohydrate Interaction in a Living Cell

“…[18,19] In molecular recognition force spectroscopy measurements, the AFM tip is upgraded to a sensor and is functionalized with ligands by means of a specially designed coupling procedure. [11] The receptor molecule is also tethered to an ultraflat support in a similar way. Alternatively, the receptor molecule may be present on the surface of a living cell or an organelle.…”

“…[9] Atomic force microscopy (AFM) based molecular recognition force spectroscopy has emerged as a powerful technique for detecting forces and the dynamic processes of biological interactions at the single-molecule level, owing to its capability of measuring forces in the piconewton range under physiological conditions. [10][11][12][13][14][15][16][17] Molecular recognition force spectroscopy provides new insights into the dynamics of recognition processes and interaction energy landscapes. [18,19] In molecular recognition force spectroscopy measurements, the AFM tip is upgraded to a sensor and is functionalized with ligands by means of a specially designed coupling procedure.…”

Beta-Actin is a Target for Transglutaminase Activity at Synaptic Endings in Chicken Telencephalic Cell Cultures

“…Inhibition of Microtubule-polymerisation was reported to disrupt cadherin adhesion in epithelial cells, but this effect was only observed after 1-2 h of treatment with nocodazole and was most likely indirect, due to disrupted trafficking of adherens junctions proteins (Waterman-Storer et al 2000). Also the depolymerisation of microtubules has no direct effect on VE-Cadherin clustering in endothelial cells (Chtcheglova et al 2010). Therefore, we do not expect a direct cadherin adhesion influencing role for microtubules in our system.…”