Figure 1.
Schematic descriptions of membrane tether formation and biofunctionalization for the AFM cantilever tip.
Membrane tether formation mediated by sLex-selectin bonding during a monocyte rolling on the endothelial layer (upper) and the strategy using AFM cantilever tips bio-functionalized by sLex to characterize the mechanics of membrane tether adhesion (lower). (modified from Yves F. Dufrêne, 2008).
Figure 2.
Typical force curves obtained from AFM measurement.
(A) Approach and retraction force curves with adhesion; and (B) without adhesion. The cantilever approaches (a to b), touches (b), makes indentation (b to c) and retracts (d) from the cell. Force of membrane tether formation (Fmtf) was measured at the sudden drop of force when a rupture of a membrane tether occurred (denoted by an arrow).
Figure 3.
Effects of Aβ, histamine, and lovastatin on P-selectin expression at the CEC surface.
(A) Fluorescent micrographs of fluorescently-labeled P-selectin at the bEnd3 cells. (B) Relative P-selectin intensity at the bEnd3 cell surface and (C) the human primary CEC surface. ***p≤0.001, **p≤0.01 compare to the control; °°° p≤0.001 compare to the Aβ (1 µM) treatment group.
Figure 4.
Effects of Aβ, and histamine on actin polymerization in CECs.
(A) Fluorescent micrographs of Oregon-green phalloidin-labeled F-actin in bEnd3 cells. (B) Relative F-actin intensity in bEnd3 cells and (C) primary human CECs. ***p≤0.001, ** p≤0.01, * p≤0.05 compare to the control; °°° p≤0.001 compare to the Aβ (1 µM) treatment group.
Figure 5.
Effects of Aβ, histamine, and lovastatin on adhesion probability at the bEND3 cell surface with AFM cantilever tips biofunctionalized by conjugating sLex at their surface.
(A) Adhesion probability was measured for cells treated with histamine, Aβ, lovastatin and Aβ and lovastatin alone. Adhesion probability was calculated by normalizing the number of force curves with adhesion events by the total number of force curves. ***p≤0.001, **p≤0.01 compare to the control; °°°p≤0.001 compare to the Aβ (1 µM) treatment group. (B) bEND3 was treated with histamine, and adhesion probability was measured. A highest adhesion probability was obtained for the cantilever coated with sLex; whereas lower adhesion probabilities were obtained for the cantilever coated with biotin only, and cells treated with antibody of P-selectin, indicating that Fmtf measured in this study are highly molecularly specific through sLex-P-selectin bonding. ***p≤0.001compare to the sLex coating group.
Figure 6.
(A) Cell stiffness (Elastic modulus) for bEND3 cells treated with histamine, Aβ, lovastatin and Aβ, lovastatin alone, and latrunculin A. The elastic modulus was calculated by fitting the cell indentation part of the force curves with Hertz model. ***p≤0.001, **p≤0.01 compare to the control; °°p≤0.01compare to the Aβ (1 µM) treatment group.
Figure 7.
Aβ on force of membrane tether formation (Fmtf) mediated by sLex-selectin bonding.
(A) Fmtf was measured at the sudden drop of force when a rupture of a membrane tether occurred (Fig. 2A). A bar graph summarizes Fmtf measured for cells treated with histamine, Aβ, lovastatin and latrunculin A. ***p≤0.001, **p≤0.01, *p≤0.05 compare to the control; °°p≤0.01 compare to the Aβ (1 µM) treatment group. (B) The distributions of Fmtf were plotted for different experimental groups. Fmtf distribution for the control group is represented in unfilled bars, and superimposed with other experimental groups represented in grey bars for comparison.
Figure 8.
(A) Membrane stiffness for cells treated with histamine, Aβ, lovastatin and Aβ, lovastatin alone, and latrunculin A. (B) Membrane stiffness is measured by calculating the slope (denoted by the arrow) from 5 nm indentation at the cell surface. ***p≤0.001, **p≤0.01 compare to the control; °°p≤0.01, °°°p≤0.001 compare to the Aβ (1 µM) treatment group.