It is well known that the plasma membrane and actin cytoskeleton are intimately connected, and that their association may occur in membrane microdomains enriched in cholesterol and sphingolipids, known as lipid rafts. Cytoskeletonassociated or cytoskeleton proteins such as actin, tubulin, myosin, a-actinin and supervillin form clusters and bind to membrane microdomains. Membrane rafts also concentrate certain integrins which regulate cytoskeleton organization, membrane trafficking and connections with the extracellular matrix through focal adhesion points. Additionally, it has also been shown that cholesterol extraction by cyclodextrin treatment alters the actin cytoskeleton organization with the formation of stress fibers. In this latter study, performed with immortalized osteoblasts, the authors showed that serum deprivation followed by 60 minutes cholesterol sequestration with 5 mM MbCD caused stress fiber formation through Rho activation. In agreement with this study, using a mouse fibroblast cell line we also found that cholesterol Ginsenoside-Ro removal from plasma membrane induces actin rearrangements as well as Rho activation. However, we observed that cholesterol removal itself, in the absence of previous serum deprivation, was sufficient to produce these effects. Most importantly, we have shown that actin remodeling upon MbCD treatment occurs not exclusively due to F-actin rearrangement, but through de novo polymerization of actin filaments. Treatment with MbCD after treatment with Latrunculin A, a drug that depolymerizes the actin cytoskeleton, was capable to restore cellular actin filaments. We found that these changes in the actin cytoskeleton reflected on the cellular mechanical properties, with MbCD treated cells becoming stiffer when compared to control non-treated fibroblasts. In 
fact, it was previously demonstrated that both surface tension s and bending modulus k can considerably vary not only between different cell types but also among the same cells that are performing different functions. Under all these conditions, changes in the cytoskeleton, as well as in its interaction with the plasma membrane can support these results. This seems also to be the case for our findings in this paper. Tether extraction experiments using optical tweezers in control and cholesterol sequestered fibroblasts clearly showed that the surface tension s, increases significantly in cells treated with the cyclodextrin. Moreover, we used for the first time the defocusing microscopy technique to study time-dependent effects of cholesterol removal from cells. We showed that the relaxation time t significantly increases in the beginning of MbCD treatment and then returns to lower values, more similar to controls, at the end of the treatment. The increase in relaxation time at the beginning of cholesterol sequestration may be explained by an enhancement in cell viscosity due to the cytoskeleton rearrangement that occurs during that period. With this methodology we were also able to obtain information on membrane fluctuation amplitude, and we showed that during cholesterol removal the amplitude Cinoxacin decreased, indicating that membrane-cytoskeleton fluctuation become less prominent upon cholesterol sequestration. This time-dependent analysis of cholesterol removal provided by DM is a very important result since we showed, through this technique, for the first time, the kinetic behavior of cholesterol extraction from cell plasma membrane. Using this technique we showed that the effect of cholesterol removal on cellular mechanics occurs very fast, in the first 10 minutes of exposure to the drug. Also, the bending modulus values increased when cells had less amount of cholesterol in their membranes.