Figure 5. Time Course of Localization of CO Proteins at IR-Induced Recombination Sites Immunolocalization of CO proteins (GFP::COSA-1, MSH-5, and/or ZHP-3) in pachytene nuclei from gfp::
cosa-1;
spo-11 worms, either in the absence of IR (A, left) or at the indicated times following exposure to 1 kRad IR. Scale bars, 5 mm. (A) Localization of COSA-1 and ZHP-3 or MSH-5 in late-pachytene nuclei in the absence of IR (pre-IR) and 8 hr post-IR. In the unirradiated
spo-11 control, ZHP-3 persists along the lengths of the chromosomes, and the majority of nuclei lack COSA-1 and MSH-5 foci; a subset of nuclei have one or two COSA-1/MSH-5 aggregates (indicated by asterisks). 8 hr post-IR: six bright COSA-1 foci localize at the heads of comet-like ZHP-3 signals. (B) Mid-to-late pachytene region of a 1 hr post-IR germline. Abundant IR-induced MSH-5 foci are detected specifically in midpachytene nuclei (left), whereas MSH-5 foci are not detected above baseline in late-pachytene nuclei (right; 0, 1, or 2MSH-5 signals colocalize with COSA-1, as in unirradiated controls). (C) GFP::COSA-1 localization in nuclei within the late-pachytene region at 2.5 and 4 hr post-IR; fields also include a few midpachytene nuclei (at the left) and a few early diplotene nuclei (at the right). Circles indicate nuclei in which six COSA-1 foci are detected. At 2.5 hr post-IR, nuclei with six COSA-1 foci are limited to a narrow zone near the start of the latepachytene region. At 4 hr post-IR, the zone of nuclei with six COSA-1 foci has expanded, presumably reflecting movement into and progression through late pachytene of nuclei that had been exposed to IR during midpachytene. (D) Localization of MSH-5 and COSA-1 at 8 hr post- IR in a region spanning the mid-to-late pachytene transition. (Left inset) Midpachytene nuclei, showing MSH-5-only foci, in excess of eventual COs. (Right) Late-pachytene nuclei, showing six MSH-5 foci that colocalize with six COSA-1 foci. See also Figure S5.