de novo at the interface of homologous axes that have progressed to late prophase without SC (Figure S2). To explore the dynamics of the full-length SC in budding yeast, we next created a second ndt80 diploid strain, K39, in which one ZIP1 locus encodes a tagged version of ZIP1 (ZIP1-GFP) under GAL1 promoter control, while the other ZIP1 locus is unmodified. After 24 hours of sporulation, (82%, n = 148) of K39 meiotic nuclei exhibited full length SCs comprised of untagged Zip1 (as judged by the near absence of GFP on spread meiotic chromosomes). We used this K39 strain to ask: Will new Zip1 subunits continue to incorporate into pre-existing full-length SC structures, or will such superfluous Zip1 subunits become sequestered into a polycomplex structure? (Figure 1A). K39 meiotic cells that had been sporulating for 26 hours (and thus predominantly containing full-length SC) were exposed to b- estradiol to induce the expression of Zip1-GFP (Figure 1B). Surface spread nuclei from uninduced and induced meiotic cells harvested 1, 2 or 3 hours after b-estradiol addition were immunostained with anti-GFP and anti-Zip1 antisera to monitor the distribution of newly induced Zip1-GFP subunits, relative to previously assembled SC. Our induction experiment revealed that new Zip1-GFP readily incorporates into previously established, full length SCs instead of forming polycomplex (Figure 1C, 1D). 96% (n = 25) of cells harvested after 1 hour of incubation with b-estradiol exhibited Zip1-GFP incorporation along the length of fully formed SC. Of these nuclei, none exhibited polycomplex. Longer incubation in b- estradiol correlates with increasing levels of Zip1-GFP incorpora- tion: over half of nuclei monitored after 2 hours of induction exhibited Zip1-GFP completely coincident with unlabeled Zip1 along extensive lengths of SC (Figure 1D). Uninduced control nuclei exhibited rare GFP staining, resulting from either a low level of constitutive expression allowed by the inducible promoter system in some cells, or from nonspecific background staining. When we examined Zip1-GFP induction in meiotic mutants, such as zip3, that display polycomplex in addition to SC stretches, induced nuclei always exhibited Zip1-GFP incorporation into polycomplex (as well as into SC stretches, see below). Post-Synapsis Incorporation of Wild-Type Zip1 Rescues the Meiotic Arrest Associated with Zip1-4LA SC To explore whether the newly induced Zip1-GFP that decorates pre-established SCs is functionally incorporated, we took advan- tage of a previously characterized zip1 allele, zip1-4LA [29]. While zip1 mutant cells (of the BR1919 genetic background) fail to build SC but do sporulate at reduced levels, zip1-4LA homozygotes build full-length SC, exhibit a normal level of crossover recombination but altogether fail to make spores. Instead, zip1- 4LA meiotic cells arrest late in prophase with fully synapsed chromosomes (thus zip1-4LA meiotic cells cytologically resemble ndt80 meiotic cells at late prophase). Mitra and Roeder (2007) previously suggested that the pachytene arrest exhibited by zip1- 4LA cells is triggered by an assembled (albeit defective) SC, based on their observation that spo11 zip1-4LA double mutants, in which Zip1-4LA is expressed but fails to assemble SC, sporulate to the same extent as spo11 single mutants. We reasoned that if Zip1 subunits functionally incorporate into previously deposited, full- length SC, then post-synapsis expression of wild-type Zip1 (or Zip1-GFP) may be sufficient to ‘‘remodel’’ SCs initially built of Zip1-4LA protein and suppress the meiotic arrest associated with Zip1-4LA SCs. To examine the possibility that new incorporation of Zip1 can remodel and alter the behavior of a full-length SC, we built a strain in which one chromosomal ZIP1 locus carries PGAL1[ZIP1] SC Dynamics at Steady State or PGAL1[ZIP1-GFP] while the other chromosome contains the zip1-4LA allele under the endogenous ZIP1 promoter (Figure 2A). After 24 hours in sporulation media, over 90% of meiotic nuclei from this strain exhibited late prophase chromosome morphology and full length SCs (built of Zip1-4LA protein), with little GFP visible in those meiotic nuclei carrying the PGAL1 [ZIP1-GFP] cassette (Figure 2B). Moreover, less than 1% of these cells eventually formed spores or spore-like structures after 40 hours in sporulation media (Figure 2C). When b-estradiol was added to the PGAL1[ZIP1-GFP] - containing strain at the 24 hour time point, Zip1-GFP was readily detected in full-length SCs after just one hour of induction (Figure 2B). Thus, SCs built of Zip1-4LA are capable of incorporating Zip1-GFP subunits. Moreover, cells from strains containing either the ZIP1 or ZIP1-GFP inducible allele that were sporulated in the presence of b-estradiol exhibited near wild-type sporulation efficiency (Figure 2C). These results indicate that post-synapsis incorporation of Zip1 can functionally alter SC behavior. Sites of Initial Zip1 Incorporation into Previously Established SC Localize near a Synapsis Initiation Protein Early Zip1-GFP incorporation into previously established, full- length SC exhibits a non-uniform pattern (Figure 1C, Figure 2B). Instead of a uniform appearance throughout the SC surface, discrete Zip1-GFP foci initially decorate the previously established SC. This pattern suggests the existence of discrete sites along the length of the SC where Zip1 incorporation is favored. As SC normally builds from multiple discrete sites along the length of chromosomes during its assembly, we asked whether such sites of initial Zip1-GFP entry exhibit characteristics of synapsis initiation sites. We first assessed whether sites of initial Zip1-GFP incorporation into full-length SCs co-localize with the Synapsis Initiation Component (SIC), Zip3 [16]. Meiotic nuclei containing fully- synapsed chromosomes and expressing Zip3-MYC were exposed to a short period (approximately 459) of ZIP1-GFP expression before they were harvested for chromosome spreads and immunostained with anti-Zip1, anti-GFP and anti-MYC antibod- ies. Zip1-GFP and Zip3-MYC distribution was assessed on SCs from nuclei with maximally spread chromosomes. 72% (n = 406) of total Zip1-GFP foci on chromosomes localized directly adjacent to or overlapping (most typical) a Zip3-MYC focus, and 93% (n = 122) of total Zip1-GFP short linear stretches partially overlapped a Zip3-MYC focus (Figure 3). Zip1-GFP induction does not itself cause recruitment of additional Zip3-MYC to chromosomes, as meiotic chromosomes exhibit an equal number of Zip3-MYC foci before and after ZIP1-GFP induction (Figure 3C). In order to assess whether the localization of initial post-synapsis Zip1-GFP incorporation events near Zip3-MYC foci is significant, Monte Carlo sampling analyses with 106 iterations (see Methods) were performed on a subset (n = 69) of well-spread Zip1 stretches that exhibited a range of the smallest GFP foci (0.58 mm or less; average size = 0.31 mm), representing the earliest Zip1-GFP incorporation events (see Figure S6 for examples). Results from Monte Carlo simulations indicated that the observed frequency of post-synapsis Zip1-GFP incorporation events that are completely encompassed by a Zip3-MYC focus (42/117) is significantly higher than expected from a random distribution of Zip1-GFP foci on Zip1 stretches, given the spatial organization of Zip3-MYC foci on each stretch and the dimensions of Zip1-GFP foci randomly taken from the sampled Zip1-GFP population (P = 0.0072). Further- more, this statistical test indicated that the observed frequency of post-synapsis Zip1-GFP positioned directly adjacent to, partially PLOS Genetics | www.plosgenetics.org 3 October 2012 | Volume 8 | Issue 10 | e1002993