SC Dynamics at Steady State exhibits dotty to linear decoration of SCs in induced nuclei. Scale, 1 mm. Scatterplots in (D) display the number of Zip1-GFP stretches that are 0.35 mm or longer associated with full-length SCs per nucleus (left) or the cumulative length of Zip1-GFP per nucleus (right) after various periods of ZIP1-GFP induction. Circles indicate values for individual nuclei; horizontal lines and error bars represent the mean and standard error of the mean, respectively. doi:10.1371/journal.pgen.1002993.g001 overlapping with or completely encompassed by a Zip3-MYC focus (78/117 for this dataset) is significantly higher than expected from a random distribution (P = 0.008). We conclude that at least a subset of initial post-synapsis Zip1-GFP events preferentially colocalize with Zip3 foci. During wild-type meiotic prophase in the BR1919 strain background, 50–80% of detectable earliest synapsis initiation events occur at centromeres [19]. We assessed whether ‘‘post-synapsis’’ Zip1 incorporation usually occurs first at centromeres by monitor- ing the distribution of induced Zip1-GFP relative to a tagged centromere protein, Ctf19-MYC (Figure 4). After approximately 459 of induction, nuclei with earliest Zip1-GFP incorporation events (i.e. nuclei with between 4–12 short GFP stretches) were examined by immunostaining. Among these nuclei, typically fewer than half of short (0.35–0.5 mm) Zip1-GFP entities were localized at or adjacent to a centromere, indicating that centromeres are not completely occupied by Zip1-GFP prior to Zip1-GFP incorporation at chromosome arm sites (Figure 4B). We next examined all centromeres in a population of nuclei exhibiting clear Zip1-GFP induction (.12 discrete Zip1-GFP incorporation events per nucleus). Among centromeres in this population, 28% (n = 364) colocalized with a Zip1-GFP focus, 42% colocalized with a short or Figure 2. Post-synapsis incorporation of Zip1 or Zip1-GFP suppresses sporulation arrest triggered by SC built with Zip1-4LA. (A) Cartoon outlines the induction experiment carried out using K29 and K30. Meiotic cells expressing Zip1-4LA, instead of Zip1, arrest in late prophase with full-length SCs. At 24 hours of sporulation, most zip1-4LA/PGAL1[ZIP1-GFP] GAL4-ER/+ meiotic prophase-arrested cells exhibit full-length SC. ZIP1 or ZIP1-GFP expression was induced in such cells at 24 hours, and then either processed for chromosome spreads (at 1 hour post-induction) or scored for spore formation (at 16 hours post-induction). (B) Nuclei from uninduced (upper panels) and induced (two examples, lower panels) K30 cells, were surface-spread and labeled with DAPI (white), anti-Zip1 (red), anti-GFP (green) following 1 hour of ZIP1-GFP induction; lower panels show that SC built with Zip1-4LA readily incorporates induced Zip1-GFP. Scale, 1 mm. (C) Bar graph depicting quantitation of spore production by uninduced cells expressing only Zip1-4LA and following induction of Zip1 or Zip1-GFP. Cells containing PGAL1[ZIP1] or PGAL1[ZIP1-GFP] and GAL4-ER were exposed to b–estradiol for indicated time periods before washing in sporulation media; spore formation was assayed approximately 16 hours later. While uninduced cells produced less than 0.5% spores, cells exposed to induced Zip1 or Zip1-GFP after 24 hours of sporulation produced between 30–50% spores, which is near wild-type for this BR1919 strain background. 1000 meiotic cells were assayed for each strain at each time point; shown are data from three independent experiments. doi:10.1371/journal.pgen.1002993.g002 PLOS Genetics | www.plosgenetics.org 5 October 2012 | Volume 8 | Issue 10 | e1002993