Figure 6. Mitotic spindle defects in slx5Δ and slx8Δ mutants.

(A) Time-lapse video microscopy of wt, slx5Δ and slx8Δ cells expressing GFP-Tub1. The upper panel shows a metaphase spindle in a wt cell at 2 minute intervals. Spindle elongation is initiated at t = 18′. The two panels below show examples of aberrant positioning of metaphase spindles in slx5Δ and slx8Δ cells during a temporary metaphase arrest. Arrows indicate spindle dislocation from the bud neck. Contours of cells are marked with a white line and are based on the DIC image. Scale bars, 5 µm. (B) Example of a spindle in slx8Δ, followed from metaphase to late anaphase. Colours are inverted to increase visibility of the astral microtubules at the outer tips of the spindle. The cell has a prolonged metaphase (t = 0′–52′) during which the spindle dislocates into the bud (t = 4′). Entry into anaphase is initiated at t = 52′, followed by spindle extension (t = 54′–80′). Formation of a fish hook spindle is apparent during late anaphase (t = 76′–80′). Scale bar, 5 µm. (C) Examples of fish hook spindles in slx5Δ and slx8Δ and a normal elongated spindle in wt during late anaphase. Scale bars, 5 µm. (D) Benomyl sensitivity assay. Growth rate of yeast cells is measured on YPD plates complemented with benomyl or DMSO (control). Images are after two days growth at 30°C. The benomyl-sensitive SAC mutant mad2Δ is included as control. (E) Quantification of spindle length, defined as the distance between two spindle pole bodies in wt, slx5Δ and slx8Δ expressing Spc42-GFP. Spindle length (n >100) was quantified from 20 minutes before anaphase onset to completion of anaphase. Grey lines depict the spindle length of individual cells. Black lines represent the average wt spindle length ± s.d., which is also shown as reference in the slx5Δ and slx8Δ plots. (F) Quantifications of spindle phenotypes in wt, slx5Δ and slx8Δ during metaphase and anaphase.