SC Dynamics at Steady State 22 hours of sporulation (when most nuclei contain full-length SC). SC intrinsic fluorescence was measured in three adjacent 0.066 mm3 ROI volumes, placed over well-spread regions of SCs in multiple nuclei for each time point. Circles indicate values for individual ROI volumes; each time point contains 120 circles. Average fluorescence intensity of Zip1-YFP increased in the strain where ZIP1-YFP is induced (blue, left graph), while fluorescence intensity remained similar between time points when untagged ZIP1 is induced (blue, right graph). While several time points exhibited ranges that were significantly different from the earliest (23 hr) time point (for both uninduced and induced columns), only the 26 hr and 28 hr induced columns from the left graph display a two-tailed P value of ,0.0001 (Mann-Whitney test). Horizontal bars indicate the mean. (C) Bar graphs show quantitation of Zip1 levels in SC, measured by intensity of anti-Zip1 antibody, from both strains in uninduced and induced conditions. SC growth in induced strains from both experiments increased to a similar extent despite whether Zip1-YFP (left) or Zip1 (right) was induced. As optimal exposure time of labeled Zip1 is indirectly proportional to the level of Zip1 in SCs, 1 divided by the average exposure time is shown. Optimal exposure times that maximize dynamic range were selected by the Softworx application in each case. Error bars indicate the standard error of the mean (n = 10). For (B) and (C), similar results were obtained from 2 independent time course experiments (one experiment is displayed here). doi:10.1371/journal.pgen.1002993.g006 Zip3-MYC foci localize to Zip1‘‘valleys’’ as well as to Zip1 ‘‘peaks’’ (Figure S3) and that meiotic cells missing Pch2, a protein that promotes the non-uniform distribution of Zip1 in SC [30], nevertheless exhibit a non-uniform spatial pattern of Zip1-GFP deposition into previously established SCs that looks indistinguish- able from PCH2+ cells (see below). SIC Components, Pph3, and Pch2 Are Dispensable for a Non-Uniform Pattern of Initial Zip1 Incorporation into SC Our observation that most initial sites of Zip1-GFP incorpora- tion occur at or near Zip3 foci on full-length SCs, in conjunction with previous demonstrations that the SICs, Zip2, Zip3 and Zip4, co-localize on fully synapsed chromosomes, raises the possibility that post-synapsis Zip1 incorporation requires SIC function. It is technically challenging to address the question of whether SIC function is required for SC maintenance dynamics, since SIC proteins are normally required for SC assembly in the first place. However, while SIC proteins are indispensable for SC assembly at a set of recombination sites along chromosome arms, SC assembly at centromeric synapsis initiation sites is less reliant on SIC function, provided that the Fpr3 and Zip3 proteins are absent [5]. In fact, zip3 mutant meiotic nuclei assemble a limited amount of SC, using predominantly centromeric synapsis initiation sites, even when Fpr3 is present [16,19]. Thus we monitored initial Zip1- GFP incorporation into previously established SC in zip3 single mutants, as well as in meiotic nuclei from zip3 fpr3, zip2 zip3 fpr3 and zip4 zip3 fpr3 mutant strains. After a short incubation in b-estradiol (45 minutes), 62%, (n = 1054) of Zip1 stretches exhibited by SIC-defective meiotic nuclei exhibited Zip1-GFP incorporation at multiple discrete sites across their entire lengths, suggesting that new Zip1 incorporation into previously established SCs can occur independent of SIC activity per se. Moreover, while SIC-defective mutants exhibited a lower cumulative length of SC as compared to wild type meiotic pachytene nuclei (Figure S4), they exhibited a similar average extent of incorporation of Zip1-GFP per unit length of previously established SC (Figure 5B and Figure S4). While de novo synapsis initiation occurs predominantly at centromeric sites in SIC- deficient meiotic cells [5], chromosomal arms exhibit no significant deficit in favored sites of post-synapsis Zip1-GFP incorporation, as seen by the existence of a centromere marker in our immuno- stained preparations (Figure 5A) and consistent with our measurements above. We note that strains missing Zip2 and Zip4 exhibited a slight reduction in the frequency of discrete Zip1- GFP incorporation events per cumulative length of SC (Figure S4). However the range in the fraction of SC decorated by Zip1-GFP after a 45-minute induction in Zip2-and Zip4-deficient strains (data derived from the same experiments) was similar to wild type (Figure 5B). These observations are consistent with the idea that Zip1-GFP incorporation perhaps occurred slightly faster in Zip2 and Zip4-deficient strains during these experiments, with either a similar or a reduced number of favored sites. Overall, our analyses demonstrate that while post-synapsis Zip1-GFP incorporation initially often localizes adjacent to Zip3, (and by inference, Zip2 and Zip4), SICs are not required, per se, for ongoing Zip1 incorporation into previously established SC, nor for the existence of discrete sites of initial Zip1 incorporation. The Pph3 phosphatase regulates Zip1 phosphorylation status during meiotic prophase [31]. In light of the known role for phosphorylation dynamics in regulating assembly and disassembly dynamics of intermediate filaments [32,33], we were interested in whether pph3 mutant cells would exhibit a defect in post-synapsis Zip1-GFP incorporation. The pattern and extent of Zip1-GFP incorporation into full length SCs built in the absence of Pph3 is similar to that displayed by wild-type cells, as shown in Figure 5B. Similarly, SCs built in the absence of the Pch2 protein, which has been proposed to influence the distribution of Zip1 within the SC central region [30] exhibits a non-uniform spatial pattern of Zip1- GFP deposition into previously-established SCs that appears similar to wild-type meiotic nuclei (Figure 5B). A Non-Uniform Spatial Pattern of Initial Zip1 Incorporation into Previously Established SC Occurs in the Absence of a Homolog Since Zip3 are presumed to mark interhomolog crossover recombination intermediates, we asked whether post-synapsis Zip1-GFP incorporation dynamics depend upon an interaction with a homolog by examining Zip1-GFP incorporation into assembled SC during haploid meiosis. MATa/MATa haploid cells carrying an untagged ZIP1 gene on a CEN plasmid and an inducible ZIP1-GFP gene at the chromosomal locus were sporulated for 26 hours and then exposed to b-estradiol to induce ZIP1-GFP expression. A fraction of surface-spread nuclei from sporulated haploid cells exhibited extensive Zip1 assembly, albeit with a temporal delay (18/101 nuclei exhibited long Zip1 stretches and 12/101 nuclei displayed Zip1 assembled along the full length of all chromosomes). Although SC assembly in haploid meiotic cells has been reported previously [34], it should be noted that the ndt80 mutation in our strains may account for the somewhat higher frequency of full-length Zip1/SC stretches observed. After a 45-minute induction, 100% (n = 25) of those haploid meiotic cells containing extensive Zip1 stretches displayed Zip1-GFP deposition at multiple discrete sites along the length of SCs (Figure 5A). Thus, a homolog is dispensable for the establishment of an SC architecture that displays a non-uniform pattern of initial Zip1 entry sites. Interestingly, our studies revealed that Zip1 assembled on haploid chromosomes also exhibits a focal pattern of Zip3 (Figure S5), suggesting the possibility that Zip3-marked recombination structures (presumably involving sister chromatids) exist in haploid meiotic nuclei. Zip1 Subunit Loss from Previously Assembled SC In order to investigate whether Zip1 subunits exit to the same extent as they enter the SC structure, we carried out a PLOS Genetics | www.plosgenetics.org 10 October 2012 | Volume 8 | Issue 10 | e1002993