For each gonad, foci were counted in 20 non-overlapping nuclei in the 2C3 rows prior to the 1st row in which a past due pachytene nucleus was observed
For each gonad, foci were counted in 20 non-overlapping nuclei in the 2C3 rows prior to the 1st row in which a past due pachytene nucleus was observed. and end result of restoration at CO and non-CO sites. system to investigate how meiotic DNA restoration proteins and meiotic chromosome structure collaborate to bring about a robust end result of meiosis, meiosis We re-examined the dynamic localization of multiple DSB restoration proteins at the sites of ongoing recombination during the course of meiotic prophase, using a nuclear distributing protocol that enables improved visualization of these events (Loidl et al., 1991; Pattabiraman et al., 2017). Chromosome axis parts and chromosome-bound restoration proteins are retained in these preparations, but nucleoplasmic protein swimming pools that obscure recombination foci are washed out, resulting in superior detection relative to previously-used methods (Fig S1A). At the same time, the relative spatial corporation of nuclei within a gonad is definitely maintained largely undamaged, conserving the spatial/temporal gradient of nuclei progressing through meiotic prophase. Fig 1A and Fig S1B display simultaneous visualization of RAD-51, the sole recombinase, and RPA-1, a subunit of the eukaryotic ssDNA binding protein RPA. Much like previous Nomegestrol acetate reports (Alpi Nomegestrol acetate et al., 2003; Colaiacovo et al., 2003), RAD-51 foci are recognized beginning in zygotene, rise in abundance and reach a plateau during early pachytene before declining and then disappearing during late pachytene (recognized based on loss of the early prophase marker DSB-2 (Fig S1B; (Rosu et al., 2013)). RPA foci Hyal2 in the beginning rise in abundance in parallel with RAD-51, but RPA foci continue to increase in large quantity after RAD-51 foci have peaked and begun to decrease, and they accumulate to much higher levels, plateauing at 30 4 foci per nucleus (Fig 1B and S2C). RPA foci decrease markedly in the early-to-late pachytene transition, but RPA remains detectable at a substantial quantity of sites before eventually being lost from all sites in late pachytene. Open in a separate windowpane Fig. 1 Localization of RAD-51, RPA, BLM, MSH-5 and COSA-1 during meiotic prophase (observe also Figs S1CS2)(A) Top: Representative images of meiotic phases in a spread WT gonad (also observe Fig S1B). DSB-2 shows nuclei in early prophase, and HTP-3 marks chromosome axes. (B) Top: Representative nuclei from a spread gonad. (A and B) Bottom: Quantitative displays of appearance, disappearance and co-localization of indicated foci in one gonad (unique from the good examples depicted). (C) Top: Image of the early-to-late pachytene transition region of a spread gonad, with individual nuclei displayed by white circles. Bottom: Quantitative display of BLM, MSH-5 and late COSA-1 foci in the portion of the gonad demonstrated above. (D) Remaining: WT gonad expressing GFP::COSA-1. Right: enlargement of the early (red format) to-late (green) pachytene transition, coinciding with the appearance of six late COSA-1 foci. Level bars (in ACD): 5 m. (E) Schematic of appearance and disappearance of recombination factors at recombination sites during meiotic prophase. MSH-5 and MSH-4 make up the heterodimeric MutS complex (for Nomegestrol acetate review: Manhart and Alani, 2016) that concentrates at CO-designated sites in late pachytene and is essential for CO formation in (Kelly et al., 2000; Yokoo et al., 2012; Zalevsky et al., 1999). MSH-5 foci 1st appear in zygotene and maximum near the end of early pachytene at an average of 22 4 MSH-5 foci per nucleus (Fig 1B and S2C). At this stage, MSH-5 foci nearly always colocalize with RPA, with fewer than one RPA-negative MSH-5 focus per nucleus. MSH-5 foci represent interhomolog (IH) recombination intermediates, as they are usually not recognized on chromosome segments that are.