Elasticity and Adhesion Force Mapping Reveals Real-Time Clustering of Growth Factor Receptors and Associated Changes in Local Cellular Rheological Properties

Abstract

Cell surface macromolecules such as receptors and ion channels serve as the interface link between the cytoplasm and the extracellular region. Their density, distribution, and clustering are key spatial features influencing effective and proper physical and biochemical cellular responses to many regulatory signals. In this study, the effect of plasma-membrane receptor clustering on local cell mechanics was obtained from maps of interaction forces between antibody-conjugated atomic force microscope tips and a specific receptor, a vascular endothelial growth factor (VEGF) receptor. The technique allows simultaneous measurement of the real-time motion of specific macromolecules and their effect on local rheological properties like elasticity. The clustering was stimulated by online additions of VEGF, or antibody against VEGF receptors. VEGF receptors are found to concentrate toward the cell boundaries and cluster rapidly after the online additions commence. Elasticity of regions under the clusters is found to change remarkably, with order-of-magnitude stiffness reductions and fluidity increases. The local stiffness reductions are nearly proportional to receptor density and, being concentrated near the cell edges, provide a mechanism for cell growth and angiogenesis.

Nanoscale visualization and characterization of Myxococcus xanthus cells with atomic force microscopy

“…The local modulus of elasticity (Young's modulus, E), which averaged 0.25 MPa, was determined for live M. xanthus cells. This E value falls between that of yeast cells (Ϸ1 MPa) (36,41) and mammalian cells (Ϸ1-200 kPa), measured with AFM (42)(43)(44)(45). AFM measurements are innately local and deal with mechanical properties of the cell at the nanoscale.…”

“…Studies using ''bacteria threads'' have estimated E on ''wet'' Bacillus subtilis at Ϸ30 MPa (40), 100 times higher that the values we obtained with AFM on unfixed M. xanthus in aqueous conditions. AFM has been used to measure the Young's modulus on yeast cells (averaging Ϸ1 MPa) (36,41) and mammalian cells, which have highly variable and spatially dependent E, and usually falls in the 1-to 200-kPa range (42)(43)(44)(45). The E value we obtained on wild-type M. xanthus cells therefore reflects the relative stiffness of the M. xanthus cell wall as compared with other organisms.…”

Force measurements with the atomic force microscope: Technique, interpretation and applications

“…It has been applied to biological model systems as biotin-streptavidin [1189], intercellular adhesion molecule-1 (ICAM-1) and anti-ICAM-1 [1159], ferritin-anti-ferritin [1190], fibrinogen-anti-fibrinogen [1191] and tymine and adenine [1192,1193]. The obvious potential for mapping distributions of biomolecules on cell surfaces has been exploited to image distribution of mannan polymers on the yeast cells [1194], sugar chains on tissue sections of the rat vomeronasal epithelium [1195], receptor-associated protein binding proteins on 3T3 fibroblasts [1196], vitronectin receptors on a murine osteoblastic cell [1197], vascular endothelial growth factor (VEGF) receptor on bovine aortic endothelial cells [1198], tyrosine kinase A on PC 12 nerve cells [1199], calcitonin receptors on bone cells [1200]. AFM tips functionalized with Helix pomatia lectin that interacts specifically with a glycolipid on group A red blood cells allowed discrimination by adhesion maps between group A and group 0 red blood cells [1201] (see Fig.…”

Recent progress in AFM molecular recognition studies

“…So far, this method has been applied to different cell types, including red blood cells [25], osteoclasts [36], and endothelial cells [37]. The power of this approach in microbiology (bacterial pathogenesis) has also been demonstrated (Fig.…”