Figure 3.

Active force generation by myosin-V drives the cytoplasmic-skeleton out-of-equilibrium. (A) At frequencies below 400 Hz the spontaneous motion of vesicles in WT oocytes (green circles) is larger than expected for thermal equilibrium (blue circles). This shows that active forces are contributing to vesicle motion in this regime as highlighted by the red shaded region. This is direct evidence of nonequilibrium behavior in the cytoplasmic-skeleton (via violation of the FDT). At high frequencies the observed vesicle motion resembles thermal motion indicated by the blue-shaded region. (B) WT oocytes (blue) are the furthest from equilibrium as shown by their higher effective energy. In the absence of actin (red) or when myosin-V is inactivated (black) the dynamic actin-myosin-V meshwork is compromised, and oocytes have lower effective energy. Solid lines are theoretical fits (see equations in the Supporting Material; error bars = mean ± SE). (C) The cell force spectrum (S_cell) experienced by vesicles is the sum of thermal forces (S_therm) and active forces (S_active). At high frequencies S_cell (green) is dominated by S_therm (blue) and the two spectra coincide (blue shaded region). At lower frequencies S_cell is larger than S_therm showing the existence of additional active forces. Solid and dashed lines are theoretical predictions (see equations in the Supporting Material), low-frequency deviation is due to simple power-law model. Standard error of experimental data is within marker size. (D) When a representative trajectory (black) is filtered to remove the high-frequency thermal fluctuations the result is a smoothed trajectory (green) that represents actively driven motion. The difference between the true trajectory (gray) and the smoothed trajectory (green) recovers the high-frequency thermal fluctuations (blue) shown in (E). (F) The MSD of vesicles, calculated from trajectory data, indicates they undergo random-confined motion in the oocyte cytoplasmic-skeleton at short timescales. This behavior transitions to active diffusion at longer times (5), and is reminiscent of cytoplasmic stirring (33). To see this figure in color, go online.