Figure 3.

AFM indentation method for analyzing desmin IF nanomechanics in cells. (A) When indenting a cell, the AFM tip first encounters the actin cytoskeleton (blue) below the plasma membrane and then (B) the intermediate filament network (red). (C) The retracting AFM force curve specifies the cell’s response to the force F applied to indent the cell to a depth h_c. The force curve can be divided into two main segments. The lower segment corresponds to the response of the actin cytoskeleton beneath the plasma membrane, whereas the upper segment of the curve predominantly represents the response of the deeper intermediate filament network. A linear fit to the upper 50% of the force curve (red) is used to determine the elastic modulus. (D) Elastic modulus (E_s) of untransfected cells (rat-2 fibroblasts), cells transfected with WT desmin-GFP fusions (DesWT) and cells expressing two types of desmin point mutants, DesA213V, which forms filaments and DesL345P, which does not. Solid bars denote the average stiffness of the whole cell or the region away from the nucleus, and the cross-hatched bar denotes the perinuclear area. The statistical analysis shows mean values of E_s and standard deviation (* p < 0.05, ** p < 0.0001) (Plodinec et al., 2011).