SC Dynamics at Steady State Figure 7. SC builds in a Zip1 concentration-dependent manner. Scatterplot in (A) depicts the fluorescence intensity (in arbitrary units) per 0.066 mm3 (0.2560.2561 mm) ROI volume encompassing an SC segment. Circles indicate values for individual rectangular volumes, placed across the length of a well-spread SC from a fully synapsed nucleus. Three adjacent rectangular volumes were recorded per SC stretch. The X axis indicates intervals of a time course spanning 16 and 27 hours of sporulation (time points are: 16, 17, 19, 21, 23, 25, 27 hours). Two independent time courses, done via three experiments (see Methods) are plotted for each strain, containing one (blue, SM170), two (green, SM176), four (orange, SM224), or six (red, SM232), copies of ZIP1-YFP. Note that the four and six copy strains carry one or two copies of a chromosome III that carries two ZIP1-YFP tandem integration events at the LEU2 locus (determined by southern blot, see Methods). Black circles on far right indicate fluorescence intensities in polycomplex (using the same ROI volume as for SC stretches). Inset graph compares the intensities of the center Z section of an ROI volume stack from full-length versus partial SCs built in ZIP1-YFP 2 copy strains. Partial SCs were selected from incompletely synapsed, but well spread nuclei, based on comparing DAPI staining (chromosome territories) with Zip1-YFP distribution. (B) Individual bar graphs depict the extent of SC assembly in strains with a specific ZIP1-YFP copy number. For each strain at 8, 12, 16, 20 and 24 hours of sporulation, the extent of SC assembly was characterized as ‘‘Dotty’’ (Zip1 foci only, white), ‘‘Dotty Linear’’ (foci and some linear stretches of Zip1, grey) or ‘‘Full-length’’ (linear Zip1 throughout most all chromosomes, black). doi:10.1371/journal.pgen.1002993.g007 complementary version of our first Zip1 induction experiment (Figure 6A). We held ndt80 mutant cells expressing one chromo- somal copy of ZIP1-YFP (a kind gift of D. Kaback, constructed as in [35]) at late prophase arrest until a majority of nuclei exhibited full-length, intrinsically fluorescent SC central region (22 hours). Next, we induced the expression of two copies of ZIP1+ using the estrogen-inducible PGAL1 system. In addition, we carried out the same induction experiment using a strain expressing one copy of ZIP1-YFP under the endogenous ZIP1 promoter and additionally carrying two copies of ZIP1-YFP under PGAL1 control. Given the fact that Zip1 subunits rapidly incorporate into full-length SCs, we reasoned that if Zip1 exit accompanies entry, we should observe a decrease in the intrinsic fluorescence of Zip1-YFP SCs after some hours of induced Zip1 (untagged) expression. On the other hand, we should observe no such decrease in SC fluorescence in meiotic nuclei from a strain in which ZIP1-YFP is induced from two chromosomal loci. Zip1-YFP fluorescence intensity was sampled in three adjacent 0.2560.2561 mm region-of-interest (ROI) volumes (a volume that encompasses the full z-dimension of a segment of SC), positioned along the length of a well-spread SC (Figure 6). The range of fluorescence intensities of such ROI volumes did not change significantly for the uninduced strains throughout the time courses. Moreover, cells induced to express two copies of ZIP1+ exhibited no decrease in the range of SC fluorescence intensities. Interest- ingly, however, a significant increase (two-tailed P,0.0001; see Figure 6 legend) in the intrinsic fluorescence of SC volumes was observed several hours after induction of expression from two copies of ZIP1-YFP. When we quantified the intensity of Zip1 in SCs using an anti-Zip1 antibody that can detect both Zip1 and Zip1-YFP species, both strains exhibited similar, significant increases in the intensity of Zip1 immunofluorescence, presumably representing an increase in SC volume and/or density under induction conditions (Figure 6). Thus, we conclude that Zip1 subunits do not exit the SC structure to the same extent as they enter, and that, as a consequence, the full length SC continuously builds in volume and/or density during a steady state (such as meiotic prophase arrest). SC Continues to Build after Its Initial Full-Length Installation To confirm the observation that steady-state SC grows over time and to explore whether the steady-state SC has an intrinsic size constraint within the context of aligned homologs, we analyzed SC central region size during a time course of meiotic prophase in ndt80 mutant (thus prophase-arrested) meiotic cells (Figure 7A) from strains carrying ZIP1-YFP as their sole source of Zip1. We measured the intensity of Zip1-YFP in ROI volumes of full-length SC (as described above) in cells carrying one, two, four or six copies of ZIP1-YFP. All ZIP1-YFP-carrying strains exhibited similar sporulation efficiencies (data not shown) and spore viability (Table S2). Our observations from these experiments support the idea that Zip1 subunits readily enter but rarely exit the budding yeast SC. The range of intensities of Zip1-YFP within a specified ROI volume of full-length SC in all strains increased, on average, during the 11 hours of meiotic prophase sampled during the time course (Figure 7A). Importantly, full-length SCs measured at time points spanning 16–21 hours of sporulation, which likely corre- spond to periods of ‘‘normal’’ pachytene progression in our BR strain background (as opposed to an ‘‘arrested’’ pachytene state) exhibited progressively larger Zip1-YFP signals per ROI volume. Moreover, at any given time point, full-length SC from strains carrying a larger ZIP1-YFP copy number exhibited progressively larger Zip1-YFP signals per ROI volume. This increase in Zip1- YFP signal was evident even between SCs from strains carrying one versus two copies of ZIP1-YFP. Thus, even at 16 hours of sporulation, full-length SCs from strains containing two copies of ZIP1-YFP typically had experienced some measure of post- synapsis Zip1-YFP incorporation, as these SCs, on average, contain substantially more Zip1-YFP than full-length SCs from strains carrying just one copy of ZIP1-YFP (at the same early time point). These data indicate that the budding yeast SC central region exhibits net growth after its initial installation at the interface of homologous chromosomes. The fact that strains with increased ZIP1-YFP copy number displayed correspondingly increased average Zip1-YFP intensity in their full-length SCs at even the earliest time point suggests that meiotic cells carrying more copies of ZIP1-YFP generate full-length SCs earlier, on average, than cells with fewer ZIP1-YFP copies (and thus have a ‘‘head start’’ on post-synapsis incorporation of Zip1-YFP). Consistent with this idea, we observed a direct correlation between ZIP1-YFP copy number and the rate at which chromosomes achieve full synapsis (Figure 7B). (We note that these data do not indicate the mechanism underlying faster achievement of full-length SC in strains carrying additional copies of ZIP1-YFP; such a mechanism could involve additional SC initiations or faster elongation from a given initiation site.) Interestingly, Zip1-YFP polycomplex formation occurred at a frequency of 83% or more in nuclei from the ZIP1-YFP 4 and 6 copy strains, even at the earliest time points sampled, yet SC structures still exhibited net growth. This result indicates that the appearance of polycomplex per se does not signal a loss of capacity to incorporate into SC. Within the constraints of our time course experiment we did not observe any upper limit to SC intensity; in fact, during one of the two experiments shown for the strain with 6 copies of ZIP1-YFP, select SC regions exhibited intensities approaching that found in Zip1-YFP polycomplex aggregates. Together, these data suggest that a synapsed homolog pair exerts little constraint on SC growth. PLOS Genetics | www.plosgenetics.org 12 October 2012 | Volume 8 | Issue 10 | e1002993