Fig 2. SUMO associates with stabilized anaphase RCs.

(A) mel-28(RNAi) anaphase spindle, stained for DNA (blue), tubulin (green), and AIR-2 (red); AIR-2 remained RC-associated (36/46 anaphases observed) and RCs remained intact. Note that in many of these spindles AIR-2 had reloaded onto segregating chromosomes (arrowhead), potentially in preparation for later RC assembly in meiosis II. (B) Localization of SUMO (green, column 4), compared to AIR-2 (red), DNA (blue), and tubulin (green, columns 1 and 5) after mel-28(RNAi). SUMO colocalized with AIR-2 on RCs in 18/18 spindles. (C) Spindles stained for DNA (blue), tubulin (green, column 1), AIR-2 (green, column 4), and SUMO (red). Top two rows show mel-28(RNAi) in the degron::gei-17 strain without auxin. Six RCs marked by both AIR-2 and SUMO are present in both metaphase and anaphase. The bottom five rows show mel-28(RNAi) in the degron::gei-17 strain after a 30 minute auxin incubation. This condition produces metaphase spindles with six RCs (shown by AIR-2 staining), of which a varying number are SUMOylated. In anaphase, the stabilized RCs always contained SUMO. Bar = 2.5μm. (D) Quantification of part C. Percent of total RCs observed in pro/metaphase or anaphase that had either both AIR-2 and SUMO present, only AIR-2 present, or only SUMO present. (E) Quantification of part C (using the same data set as part D). Number of AIR-2 stained RCs per spindle versus the number of SUMOylated RCs during pro/metaphase or anaphase. Box represents first quartile, median, and third quartile. Lines extend to data points within 1.5 interquartile range. Note that in D and E we only quantified anaphase spindles where we could distinguish at least one intact AIR-2-marked RC, excluding spindles that progressed past the mel-28(RNAi) arrest point due to incomplete depletion.