The role of the actin cytoskeleton in the shape dependent differences in mitosis.

HeLa (YFP-paxillin) cells were cultured for 18 hours in either (A) square microwells or (B) circular microwells and imaged during interphase for actin (red) and paxillin (green); bars: 10 µm. HeLa (GFP-H2B/RFP-tubulin) cells were assessed for the parallel orientation of the mitotic spindle at metaphase after culture in (C) square or (D) circular microwells and treatment with latrunculin A for 1 hour prior to mitosis. 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. (E) The inhibition of actin polymerization in HeLa (GFP-H2B/RFP-tubulin) cells lead to increased tilting in cells cultured in the square microwells. Values represent spindle angle in degrees ± SEM. (F) Perturbation of actin polymerization in cells cultured in square microwells also increased the distribution of spindle orientation, but had little effect on cells cultured in circular microwells. (G) The perturbation of the actin cytoskeleton negated the differences in mitotic timings observed between cells cultured in square microwells (light grey bars) and cells cultured in circular microwells (dark grey bars). All values represent time in minutes ± SEM. NB = nuclear envelope breakdown, S = spindle formation, LPM = late prometaphase, M = metaphase, A = anaphase.