Mitotic Spindle Orients Perpendicular to the Forces Imposed by Dynamic Shear
Figure 6
Shear forces orient the division axis without compromising mitosis.
A: Division angle distributions for different amplitudes (vertical axis on the left) and frequencies (horizontal axis at the bottom) for RPE1 cells. At small amplitudes (γ = 10%), anisotropic division requires a very high frequency (30 Hz). At large amplitudes (γ = 100%), even very slow stimulations (0.03 Hz) suffice to align the division axis in essentially all cells. Statistical significance relative to the control was assessed using a Mann-Whitney U-test on the reflected angles | θ - 90° |. One asterisk indicates P<0.01, two asterisks indicate P<10−8. B: Similar results were obtained with the osteoblast cell line MC3T3. C: Cumulative histograms of anaphase onset times in the presence of dynamic forces for RPE1 cells. Each curve is a different experiment, performed at varying amplitudes and the same frequency 3 Hz. D: Cumulative histograms of anaphase onset times of RPE1 cells for different frequencies and constant large amplitude 100%. Strain-alignment of the division axis (which is observed for all frequencies at this amplitude) delays division only marginally. A significant delay is observed at 3 Hz; note that cell division becomes again fast at 30 Hz. E: RPE1 cell growth after force-alignment of the spindle. After each experiment, cells were collected, plated on a gridded dish (Ibidi, Munich, Germany) and the growth of individual cells was followed over three days. Note that cells are synchronized by shake-off prior to the experiment and re-seeded afterwards. After one day, cells grow exponentially. To ease comparison of the exponential growth phase between different treatments, we divided cell number by its value at day 1. For a control, we followed also the growth of cells that had been in normal culture conditions throughout (black dashed line). No significant difference in growth rate can be observed after mechanical stimulation at 0.03 Hz, 100%. Nevertheless, we observed increased cell death within the first day following stimulation at 3 Hz, 100%, probably indicating damage by the non-physiological stimulation.