taken from meiotic cultures sporulated in the presence of b- estradiol, thus clear assemblies of Zip1 are visible at the interface between aligned chromosome axes. Scale, 1 mm. (PDF) Figure S2 Late prophase meiotic axes are competent to assemble Zip1. (A) Cartoon depicts the ‘‘late Zip1 induction’’ experiment conducted. In this experiment ZIP1 expression is prevented until either 20 or 26 hours of sporulation. Homozygous PGAL1[ZIP1+] ndt80 GAL4.ER (K40), cells were sporulated in the absence of b-estradiol for 20 or 26 hours, time points during meiotic prophase arrest where the majority of ZIP1+ cells would have completed synapsis. b-estradiol was added to sporulating cultures to induce ZIP1 expression, and nuclei were surface-spread and analyzed at four successive hourly intervals following b- estradiol addition. (B) Bar graphs show that for either experiment, a substantial percentage of nuclei (n.50 for each time point) exhibited short and long Zip1 stretches assembled at the interface of aligned chromosome axes; moreover some nuclei appeared to have completed synapsis (‘‘full SC’’; red). Meiotic surface-spread chromosomes from such strains are shown in (C) labeled with anti- Red1 (red) and anti-Zip1 (green) antibodies. Scale, 1 mm. (PDF) Figure S3 Zip1-GFP incorporates into full-length SC both at sites of high and sites of low Zip1 abundance. Graph in (A) gives the number of discrete Zip1-GFP incorporation events (focus or short (0.35–0.5 mm) stretch) or Zip3-MYC foci that co-localized with an area of high Zip1 abundance (‘‘Zip1 Peak’’), low Zip1 abundance (‘‘Zip1 Valley’’), or at the terminus of a synapsed chromosome (‘‘Zip1 Termini’’) in K48. Zip1-GFP incorporation events or Zip3-MYC foci that could not be unambiguously assigned to a particular feature of the SC were grouped in the ‘‘Ambiguous’’ category. Images in (B) show examples of Zip1-GFP events (green, top two rows) or Zip3-MYC foci (green, bottom row) in Zip1 (red) valleys (arrows) or peaks (arrowheads). DNA for these nuclei is shown in white (at left). Scale, 1 mm. (PDF) Figure S4 Zip1-GFP incorporation into full length SC may be reduced in situations where the crossover: Zip1 length ratio is predicted to be low. Scatterplots in (A) show the extent of Zip1 SC present in nuclei, upon ZIP1-GFP induction (at ‘‘start’’), for the various strains involved in the incorporation experiment shown in Figure 5. Scatterplot in (B) depicts the number of discrete Zip1- GFP incorporation events (either a focus or a Zip1-GFP stretch) per cumulative length of Zip1, after a 45-minute induction of ZIP1-GFP expression. Circles indicate Zip1-GFP incorporation events per cumulative length of Zip1 for individual nuclei. Horizontal and error bars indicate mean and standard error of the mean for each column. The average number of Zip1-GFP incorporation events per mm of Zip1 is significantly reduced References 1. Bhalla N, Dernburg AF (2008) Prelude to a division. Annu Rev Cell Dev Biol 24: 397–424. 2. Zickler D, Kleckner N (1999) Meiotic chromosomes: integrating structure and function. Annu Rev Genet 33: 603–754. 3. Page SL, Hawley RS (2004) The genetics and molecular biology of the synaptonemal complex. Annu Rev Cell Dev Biol 20: 525–558. 4. MacQueen AJ, Colaiacovo MP, McDonald K, Villeneuve AM (2002) Synapsis- dependent and -independent mechanisms stabilize homolog pairing during meiotic prophase in C. elegans. Genes Dev 16: 2428–2442. 5. MacQueen AJ, Roeder GS (2009) Fpr3 and Zip3 ensure that initiation of meiotic recombination precedes chromosome synapsis in budding yeast. Curr Biol 19: 1519–1526. SC Dynamics at Steady State (compared to wild type, Mann-Whitney test) for the following strains: zip2 zip3 fpr3 (two-tailed P = 0.0005) and zip4 zip3 fpr3 (two-tailed P = 0.0032). The fpr3 strain also exhibited a significant difference from wild type (two-tailed P = 0.0113), likely due to the longer cumulative lengths of Zip1 exhibited by this single mutant (see A). (PDF) Figure S5 Zip3 foci decorate Zip1 stretches in haploid meiotic cells. Meiotic chromosomes (white) from haploid MATa/MATa cells carrying Zip3-GFP (AM2632, top row) or Zip3-MYC (K150, bottom row) were sporulated for 26 hours, and then surface spread on glass slides. Zip1 (red) and Zip3-GFP or Zip3-MYC (green) are depicted in single channels and as a merged image. Scale, 1 mm. (PDF) Figure S6 Sites of initial Zip1 entry into full-length SC localize near Zip3 foci. (A) Three surface-spread meiotic nuclei (DNA for each nucleus is shown in white, top row) from Zip3-MYC strains (K48) after a short induction of ZIP1-GFP expression (45 minutes). Zip1 staining (second row, blue) shows full length SC decorated by Zip3-MYC (red) foci. Bottom row displays several zoomed Zip1 SC stretches (blue) from each nucleus (4–5 per nucleus), which are a subset of the 69 stretches used in our statistical analysis of post- synapsis Zip1-GFP distribution relative to Zip3-MYC. Both Zip3- MYC (red) and post-synapsis Zip1-GFP incorporation events (green) are shown in zoomed images. Scale, 1 mm. (PDF) Table S1 (PDF) Strains used in this study. Table S2 Viability of spores produced by diploids carrying one to six copies of ZIP1. The far right column shows the overall spore viability for each strain. Displayed in each ‘‘Distribution of tetrad types’’ column is the frequency of tetrads containing four viable spores (4-sv), three viable spores (3-sv), two viable spores (2-sv), one viable spore (1-sv) or no viable spores (0-sv). Strain genotypes are listed in Table S1. (PDF) Acknowledgments We extend special thanks to Beth Rockmill and Scott Holmes for suggestions and critical reading of the manuscript. We thank Cassandra Johnston for assistance with protein preparation for Western blot experiments and Sarah Beattie for assistance with strain dissection. Author Contributions Conceived and designed the experiments: AJM AMV KV-M SSM. Performed the experiments: KV-M SSM AJM. Analyzed the data: KV-M SSM PL AJM. Contributed reagents/materials/analysis tools: PL. Wrote the paper: AJM. 6. MacQueen AJ, Villeneuve AM (2001) Nuclear reorganization and homologous chromosome pairing during meiotic prophase require C. elegans chk-2. Genes Dev 15: 1674–1687. 7. Zickler D (2006) From early homologue recognition to synaptonemal complex formation. Chromosoma 115: 158–174. 8. Zickler D, Lares Ld, Moreau PJF, Leblon F (1985) Defective pairing and synaptonemal complex formation in a Sordaria mutant (spo44) with a translocated segment of the nucleolar organizer. Chromosoma 92: 37–47. 9. Dong H, Roeder GS (2000) Organization of the yeast Zip1 protein within the central region of the synaptonemal complex. J Cell Biol 148: 417–426. 10. Sym M, Engebrecht J, Roeder GS (1993) Zip1 is a synaptonemal complex protein required for meiotic chromosome synapsis. Cell 72: 365–378. PLOS Genetics | www.plosgenetics.org 16 October 2012 | Volume 8 | Issue 10 | e1002993