Figure 5.
Rec8 cleavage at early prophase I exhibits defects in sister chromatid cohesion and synaptonemal complex formation. (A) Experimental regimen to express Esp1 protein during meiotic prophase I. (B and C) Analysis of sister chromatid cohesion. Strains carrying LacO array at chromosome XV and expressing Lac repressor conjugated with green fluorescence protein were analyzed for sister association in fixed whole cells (the diagram showing the location of Lac operator array at chromosome XV in B). Single culture was synchronized and divided into two SPM cultures; then Esp1 expression was induced by addition of 50 μM CuSO4 at 2 h (red arrow) in one of the two cultures. One focus indicates unreplicated chromatid or unseparated sister chromatids. Two foci indicate replicated and separated sister chromatids. (D) Esp1 overexpression in the early stages of meiotic prophase I defects in SC formation and Rec8 assembly onto chromosomes. Nuclei were immunostained with anti-HA (for Rec8-3HA) and anti-Zip1 in spread chromosomes. Representative images showing spread nuclei immunostained for Rec8-3HA and Zip1 according to four categories (category I, no signals; category II, modest number of foci; category III, large number of foci with fuzzy pattern; category IV, fully assembled lines). (E) Chromosome spreads were analyzed for the percentage of cells in Zip1 and Rec8 staining in PCup1-ESP1 strain in the presence or absence of CuSO4, according to the categories as shown in (D) (mean ± SD, n = 150–200 nuclei). (F) Representative images showing the formation of Zip1-polycomplex (PC) in PCup1-ESP1 strain in the presence or absence of CuSO4. White arrowheads indicate Zip1-PCs. (G) Quantitative analysis showing the percentages of Zip1-PCs shown in (F) (mean ± SD, n = 150–200 nuclei).