Figure 6. Analysis of Exit from Mitosis in prm3Δ heterokaryons.
(A) Cartoon of the prm3Δ heterokaryon system showing the two main classes of heterokaryons obtained. (B–F) Heterokaryons were obtained by mating cells lacking PRM3 (see Materials and methods for details). Briefly, G1 cells isolated by centrifugal, elutriation were mated at 30° for 2 hr and then loaded onto a Y04D CellASIC flow cell for imaging. Synthetic complete pH 6.0 medium was used in the flow cell during imaging and was supplemented with 100 μM IAA to induce spindle mispositioning. (B) Montages of binucleate zygotes created by mating cells lacking PRM3. Binucleate diploids are homozygous for prm3Δ and osTIR1 and heterozygous for DYN-AID, kar9Δ, mCherry-labeled Cdc3 and GFP-labeled α-tubulin (A37892 x A35570). The DIC channel was adjusted to maximize contrast. (C–D) Analysis of anaphase kinetics of cells described in Figure 6B. Class 1: n= 16. Class II: n= 21 (E) Binucleate diploids that were homozygous for prm3Δ, osTIR1, DYN-AID, kar9Δ and heterozygous for mCherry-labeled Cdc3 and GFP-labeled α-tubulin (A35570 x A35571) were analyzed for anaphase duration. Black diamonds indicated permanently arrested cells (permanently arrested ≥320 min) n=51 cells. (F) Montage of cells from Figure 6E. The DIC channel was adjusted to maximize contrast.