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First published online 5 June 2007
doi: 10.1242/jcs.001370

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The effect of cellular cholesterol on membrane-cytoskeleton adhesion

¹ Department of Physics and Astronomy, University of Missouri-Columbia, Columbia, MO 65211, USA
² Department of Biophysics, University of Pecs, H-7624, Hungary
³ Institute of Medicine and Engineering, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
⁴ Pulmonary, Critical Care and Sleep Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA
⁵ Department of Biological Sciences, University of Missouri-Columbia, Columbia, MO 65211, USA

View larger version (12K): [in this window] [in a new window]   Fig. 1. (A) A typical force vs extension curve. The dotted line corresponds to the approach curve and the solid line is the retraction curve. On the retraction curve, several step-like structures are clearly discernible, which correspond to the sequential detachment of individual tethers from the cantilever. (In principle these steps could signal the detachment of a single tether with multiple connections to the cantilever. This possibility can be excluded on the basis of the numerous earlier studies in which single tethers were extracted and no steps in the course of tether elongation were observed.) The shown retraction curve corresponds to an experiment performed with a control cell at 3 µm/second. (B) Histogram constructed from the data (size of the steps) collected in the control experiments at 3 µm/second. The solid line is the Gaussian fit to the histogram, which gives the mean value of the tether force, 33 pN.

View larger version (26K): [in this window] [in a new window]   Fig. 2. (A,C) Tether force vs tether growth velocity at room temperature (A) and 37°C (C). The solid lines are linear fits to the corresponding data sets (control, ; depletion, ; enrichment, ). Inset in A, cholesterol level measured by GLC (see Materials and Methods). ctrl, control; dpl, cholesterol depleted; enrch, cholesterol enriched. The values of slopes (eff) and intercepts (F0) are listed in Table 1. (B,D) Bar graph for F0 at room temperature (B) and 37°C (D). Error bars in A and C represent s.e. obtained from the experimental data points. Errors in the bar graphs represent the error of the linear fit to the data. All six R2 values were larger than 0.97.

View larger version (33K): [in this window] [in a new window]   Fig. 3. (A) Tether force vs tether growth velocity after latrunculin A treatment (, control; , cholesterol depleted; , cholesterol enriched). Values of the intercepts and slopes are listed in Table 1. Error bars represent s.e. (note that the error bars overlap). (B) Confocal images of the actin cytoskeleton for control (a,d), cholesterol-depleted (b,e), cholesterol-enriched (c,f) BAECs, without (top row) or with (bottom row) latrunculin A treatment. Bar, 10 µm.

View larger version (108K): [in this window] [in a new window]   Fig. 4. DIC images of cells with different cholesterol content. Cholesterol-depleted (A,D), control (B,E), cholesterol-enriched (C,F) BAECs, without (top row) or with (bottom row) latrunculin A treatment. Latrunculin-A-treated cells show a similar spherical morphology independently of cholesterol content. Bar, 10 µm.

View larger version (21K): [in this window] [in a new window]   Fig. 5. (A) A typical recovery curve in a control FRAP experiment. The fluorescence intensity is normalized to the average fluorescence intensity before photobleaching. (B) Diffusion coefficients in the various treatment cases (normalized to the value for the control). ctrl, control; dpl, cholesterol depleted; enrch, cholesterol enriched; lata, latrunculin A treated.

View larger version (8K): [in this window] [in a new window]   Fig. 6. (A) Tether length vs time, calculated using equation 2 (see Materials and Methods). The tether grows much faster after cholesterol enrichment (dotted line) compared with the control (solid line) and cholesterol-depleted (dashed line) cases. The curves for control and cholesterol depletion are similar. (B) The estimated force acting on a bond between an endothelial tether and a leukocyte. After cholesterol enrichment (dotted curve), initially, the bond force decreases much faster than in the control (solid curve) and cholesterol depleted (dashed curve) cases. The curves for the control and cholesterol depletion are again similar.

View larger version (6K): [in this window] [in a new window]   Fig. 7. The estimated membrane-cytoskeleton adhesion force, Fad, calculated using equation 1. Values of Fad for cholesterol-depleted (dpl) and cholesterol-enriched (enrch) cells are both significantly (P<0.05) different from the control (ctrl).

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