Fig. S2.
(A) Definition of the parameters used as phenomenological, model-independent indicators of intracellular rigidity. The rigidity index (RI) is defined as the area under the relaxation curve (red curve) normalized by the area under the stage displacement curve (black dashed line). The bead step amplitude Xb is defined as the initial displacement of the bead following the step displacement of the stage (Xs = 0.5 µm in all experiments except in Fig. S2 C–E). (B) Standard linear liquid (SLL) viscoelastic model composed of a dashpot of viscosity η0 in series with a Kelvin–Voigt body (spring of spring constant μ and dashpot of viscosity η in parallel). xs, xb, and x1 denote, respectively, the positions of the stage, the bead, and the junction between the spring and the Kelvin–Voigt body with respect to the center of the optical trap. The first 10 s of the relaxation curves were fitted using the model, and the rheological parameters were deduced from the fit (Materials and Methods). (C–E) To check whether the experiments were performed in a linear regime, the step displacement of the stage was varied from 0.4 to 0.6 µm. The corresponding relaxation curves of the bead position were averaged (C) and fitted using the SLL model or the PL model. The model-independent rigidity index (RI) and bead step amplitude (Xb) were measured (D and E, Top), and the spring constant and viscosity (E, Middle) and the complex shear modulus (E, Bottom) were deduced from the SLL or PL fits (n = 16, 9, and 23 for control; 0.4-, 0.5-, and 0.6-µm stage step sizes, respectively). ***P < 0.0001. (F–H) Three-dimensional localization of 2-µm-diameter beads internalized in RPE1 cells plated on crossbow-shaped micropatterns. Live cells were stained with the membrane marker CellMask and imaged using confocal microscopy. A typical example (F), the averaged x–z and y–z profiles (G, top two images) and the maximum intensity projections of the x–z and y–z profiles (G, bottom two images) are shown (from n = 10 cells). The positions of the 16 beads internalized in these cells are marked in red on the maximum intensity projections. The cell maximum height and the bead z position are quantified in H from n = 30 beads in 22 cells. (Scale bars, 5 µm.)