Dot1 is required for <i>zip1</i>-induced localization and activation of the Hop1 meiotic checkpoint adaptor.

<p>(A) Immunofluorescence of meiotic chromosome spreads stained with DAPI (blue), anti-Hop1 (green) and anti-myc (red) antibodies. Representative nuclei are shown. The same exposure time was used to capture the signal from the different strains. Spreads were prepared 24 h after meiotic induction of <i>ndt80</i> cells. Strains are: DP848 (<i>zip1</i>) and DP849 (<i>zip1 dot1</i>). (B) Quantification of the Hop1 staining pattern (left) and the number of Mek1 foci (right) on spread chromosomes analyzed as in (A). 14 and 21 nuclei were scored for <i>zip1</i> and <i>zip1 dot1</i>, respectively. (C) Representative images of <i>ndt80</i>-arrested cells expressing <i>HOP1-GFP</i> in <i>zip1</i> (DP964) and <i>zip1 dot1</i> (DP965) captured after 24 h in meiosis. (D) Quantification of the Hop1-GFP signal intensity on fluorescence images (a.u., arbitrary units). 300 individual nuclei were analyzed for each strain. (E) Dot1 is required for Hop1 phosphorylation. Western blot analysis of Hop1 in cell extracts obtained 24 h after meiotic induction in <i>ndt80</i> cells. The middle panel corresponds to an overexposure (OEx) of the blot shown in the upper panel. PGK was used as a loading control. Strains are: DP428 (<i>zip1</i>), DP655 (<i>zip1 dot1</i>), DP680 (<i>zip1 mec1</i>) and DP674 (<i>zip1 mek1</i>). Means, standard deviations and <i>P-</i>values are shown in (B) and (D).