Furthermore, our untagged Zip1 induction experiment (Figure 6) in conjunction with these growth experiments suggest that SC central region exhibits extremely low subunit turnover; previously deposited SC remains static as additional central region subunits continue to build. Discussion Zip1 Incorporates into Previously Assembled SC with a Nonuniform Spatial Pattern Our induction experiments demonstrate that the full-length SC in budding yeast is dynamic, as new transverse filament subunits (Zip1-GFP) continuously incorporate when meiotic cells are arrested at late prophase. It will be informative to learn whether additional proteins that have been implicated in generating SC central region, such as SUMO, exhibit similar post-synapsis incorporation dynamics. Instead of a uniform incorporation of Zip1-GFP along the length of a previously established SC, post-synapsis Zip1-GFP subunit incorporation initially exhibits a focal pattern. Such initial Zip1 entry sites may occur at centromeres and a majority of them localize adjacent to sites marked by the synapsis initiation component, Zip3. Our statistical analysis indicates that Zip3 marks at least a subset of initial post-synapsis Zip1-GFP incorporation sites. Our observation that at least a subset of post-synapsis Zip1-GFP incorporation events preferentially localize to Zip3-marked sites along the SC raises the possibility that many or all of the ongoing Zip1-GFP incorporation events into full-length SC could be the result of synapsis initiation activity by SIC proteins, as the de novo initiation of Zip1 assembly (synapsis) at presumed recombination sites during normal meiotic progression requires the SIC proteins Zip2, Zip3 and Zip4. On the contrary, post-synapsis Zip1 incorporation sites do not exhibit the same regulatory mechanisms as sites of de novo synapsis initiation, since our data demonstrate that post-synaptic events are not dependent on Zip2, Zip3 or Zip4 proteins (Figure 5, Figure S4). An interesting possibility is that favored Zip1 entry sites contribute to the establishment of Zip1 ‘‘peaks’’ and ‘‘valleys’’ in full length SC. However, under such a model one might expect that post-synapsis Zip1-GFP incorporation events would seldom localize to Zip1 ‘‘valleys’’. We observed that sites of post-synapsis Zip1 incorporation do not strictly correspond to either local peaks or valleys of Zip1 within the previously deposited, full-length SC, and the Pch2 protein which regulates such domains of high and low Zip1 [30] does not alter the frequency or qualitative pattern of Zip1 incorporation sites. One could reconcile our observation that a fraction of initial post-synapsis Zip1-GFP events occur at Zip1 valleys with the idea that favored Zip1 incorporation sites contribute to the formation of Zip1 peaks if we further propose that rates of Zip1-GFP incorporation varies between favored sites or at a given site over time. Alternatively, Zip1 peaks and valleys may arise from a mechanism that mediates Zip1 subunit movement laterally within the SC. Favored Zip1 entry sites are intriguing because they indicate a level of non-uniformity in SC architecture. However, with time, the pattern of post-synapsis Zip1-GFP distribution completely overlaps the full-length SC. One explanation for the progressive change in the distribution of post-synapsis Zip1 might be that the budding yeast SC maintains a capacity to add new Zip1 all along its length but certain sites are more favorable for addition than others. Alternatively, as mentioned above, perhaps the SC incorporates new transverse filament subunits exclusively at certain sites but that some newly incorporated subunits can move SC Dynamics at Steady State to a different location along the length of the SC. Indeed, the frequent occurrence of post-synapsis Zip1-GFP foci that only partially overlap Zip3-MYC gives the impression that newly incorporated subunits might grow outward from the Zip3-MYC domain. Such immunofluorescence data, however, are also consistent with a model in which post-synapsis Zip1-GFP domains grow longer via continued incorporation at both Zip3 and (eventually) non-Zip3 sites. Transverse Filaments Exhibit Robust Entry into but Minimal Exit from Full-Length SC during Pachytene Arrest Our data indicate that, once the SC has assembled along the full length of meiotic axes in cells experiencing pachytene arrest, Zip1 subunits exhibit little turnover but continue to incorporate, allowing the SC to grow to many times its original density or volume. Thus, the SC central region appears to behave more like a ‘‘tether’’ than a ‘‘treadmill’’, at least during late prophase when chromosomes are fully synapsed. The absence of significant turnover of previously deposited transverse filaments suggests that the SC may only have the capacity to remodel through new subunit addition. One can imagine that continuous SC assembly might facilitate at least some mechanistic aspects of synaptic adjustment, where an unsynapsed chromosomal domain, contiguous with an otherwise synapsed chromosome pair, eventually incorporates into the full length SC during prophase [2,36]. However, continuous assembly without SC subunit turnover raises the question of whether budding yeast can correct inappropriate synapsis. Perhaps SC assembly in budding yeast is sufficiently regulated at the outset so that irrevocable synapsis mistakes occur at an extremely low frequency. On the other hand, an SC disassembly mechanism that does not rely on ongoing central region turnover may exist that can be deployed over a local chromatin domain. Another possibility may be that SC central region can be physically disrupted, given enough applied force, without an active disassembly mechanism. Finally, the balance between subunit entry and exit may differ at different stages of budding yeast synapsis, for example during zygotene and early pachytene stages, potentially allowing error correction at those stages before solidifying a final synapsis configuration. Couteau and Zetka observed local SC central region disassem- bly in response to irradiation in C. elegans late meiotic prophase nuclei [37], suggesting that C. elegans SC may have the capacity to remodel in response to recombination intermediates. Contrary to this picture, a relatively static central region in conjunction with ongoing Zip1 incorporation preferentially at Zip3 sites raises the possibility that the budding yeast SC only structurally accommo- dates those (or a subset of those) recombination events arising prior to or concomitant with SC deposition. This possibility is consistent with several recent models for meiotic prophase chromosome dynamics in budding yeast [38,39]. Under this scenario, the budding yeast SC central region may not need to ‘‘remodel’’ to structurally accommodate recombination sites, since those that will interface with the SC will have already progressed to a certain intermediate stage prior to SC installation. Such an intermediate stage would need to involve establishing a local environment in which recombination-associated structural changes in the DNA and recombination enzyme complexes can proceed unperturbed by ongoing deposition of SC central region proteins in the surrounding vicinity. We note, however, that our observation of little global SC turnover does not rule out the possibility that SC turnover occurs differentially at distinct sites. As SC demonstrates a non-uniformity in assembly dynamics in the vicinity of PLOS Genetics | www.plosgenetics.org 13 October 2012 | Volume 8 | Issue 10 | e1002993