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. 2011 Jul 6;101(1):43-52.
doi: 10.1016/j.bpj.2011.05.044.

Cell cytoskeleton and tether extraction

B Pontes  1 , N B Viana, L T Salgado, M Farina, V Moura Neto, H M Nussenzveig
Affiliations

Cell cytoskeleton and tether extraction

B Pontes et al. Biophys J. .

Abstract

We perform a detailed investigation of the force × deformation curve in tether extraction from 3T3 cells by optical tweezers. Contrary to conventional wisdom about tethers extracted from cells, we find that actin filaments are present within them, so that a revised theory of tether pulling from cells is called for. We also measure steady and maximum tether force values significantly higher than previously published ones for 3T3 cells. Possible explanations for these differences are investigated. Further experimental support of the theory of force barriers for membrane tube extension is obtained. The potential of studies on tether pulling force × deformation for retrieving information on membrane-cytoskeleton interaction is emphasized.

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Figures

Figure 1
Figure 1
Schematic representation of the tether extraction experiment. (a) Initial equilibrium position. (b) Change in equilibrium position due to sample movement.
Figure 2
Figure 2
Tether extraction experiment in Fibroblast NIH 3T3 cell. (a) Initial situation, t = 0. (b) Final situation, showing the image of the extracted tether in bright field with ImageJ shadow north processing filter applied. Scale bar for panels a and b is 10 μm. (c) Montage of images from the video recording (see Movie S1). (d) The force curve of the tether extraction experiment, with numbers in the plot indicating the corresponding bead position in panel c; scale bar 1 μm.
Figure 3
Figure 3
Presence of F-actin inside the tether. In control cells. (a1) Actin staining (phalloidin-FITC. (a2) Zoom showing the presence of F-actin in the extracted tethers. (a3) Bright-field/fluorescence merge image from NIH 3T3 cells. (b1–b3) Same as panel a for cytoD cells. All the scale bars are 10 μm. (c) Plot representing the green-level fluorescence (GL) of each condition, as indicated in the legend, normalized by the green-level fluorescence inside the control cell (GLcontrol(cell)) (open bar).
Figure 4
Figure 4
Cell-bead immersion angle measurements using scanning electron microscopy (SEM). (a) SEM panoramic image of the entire cell showing the beads attached to its surface. Scale bar: 10 μm. (b) High magnification SEM of a bead showing the immersion angle (θ) in the cell. Scale bar: 1 μm. (c) Bright-field image of an optically trapped bead adhered to a Cytochalasin-D-treated cell. The adhesion angle measured in this case is 38° in agreement with SEM measurements. Scale bar: 10 μm. (d) Results for angle measurements in control (shaded) and Cytochalasin-D-treated cells (open). Standard errors were used as error bars. At least 20 different experiments for each situation (p > 0.1 means no significant statistical differences using the t-test).
Figure 5
Figure 5
Tether radius measured by scanning electron microscopy (SEM). (a) SEM representative image of a tether extracted from NIH 3T3 cell in control group. Scale bar: 1 μm. (b) SEM representative image of a tether extracted from Cytochalasin-D-treated NIH 3T3 cell. Scale bar: 1 μm. (c) Plot profile of the gray-level intensity in the tether for panel a. The plot profile was adjusted to half-hyperbolic tangent fits and the radius was determined as indicated in Eqs. 6 and 7. (d) Results for the tether radius (R) in control (shaded) and Cytochalasin-D-treated cells (open). At least 10 different measurements were performed for each situation (∗∗∗ means p < 0.001 in t-test statistics).
Figure 6
Figure 6
Bead coating does not alter force characteristics in Fibroblast NIH 3T3 cells. Results for maximum force values (a) and tether force values (b) using 1.52-μm radius normal and carboxylated polystyrene beads coated with different proteins as indicated in the figure legends. Standard errors were used as error bars. At least 20 different experiments for each bead-coating situation (no significant statistical difference was found for each group using the ANOVA test).
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