The role of spindle checkpoint activation in the shape dependent differences in mitosis.

HeLa (GFP-H2B/RFP-tubulin) cells were assessed for the parallel orientation of the mitotic spindle at metaphase after transfection with (A and B) scrambled siRNA or (C and D) Mad2 siRNA and culture in (A and C) square or (B and D) circular microwells to determine the role of spindle checkpoint activation. Cells cultured in circular microwells showed a random orientation of the mitotic spindle, whilst cells cultured in square microwells predominately aligned the spindle along the long axis of the cell, regardless of the depletion of Mad2. Similarly, the depletion of Mad2 had little effect on (E) the distribution and (F) average spindle orientation perpendicular to the substrate plane. Values represent spindle angle in degrees ± SEM. (G) Cells transfected with siRNA for Mad2 and cultured in circular microwells (white bars) took significantly less time to reach late prometaphase than cells transfected with control siRNA (dark grey bars), indicating that the shape dependent effect on the time required to reach late prometaphase was due to differences in spindle checkpoint activation. All values represent time in minutes ± SEM. Key: * p