Sumoylation regulates protein dynamics during meiotic chromosome segregation in C. elegans oocytes

ABSTRACT Oocyte meiotic spindles in most species lack centrosomes and the mechanisms that underlie faithful chromosome segregation in acentrosomal meiotic spindles are not well understood. In C. elegans oocytes, spindle microtubules exert a poleward force on chromosomes that is dependent on the microtubule-stabilising protein CLS-2, the orthologue of the mammalian CLASP proteins. The checkpoint kinase BUB-1 and CLS-2 localise in the central spindle and display a dynamic localisation pattern throughout anaphase, but the signals regulating their anaphase-specific localisation remains unknown. We have shown previously that SUMO regulates BUB-1 localisation during metaphase I. Here, we found that SUMO modification of BUB-1 is regulated by the SUMO E3 ligase GEI-17 and the SUMO protease ULP-1. SUMO and GEI-17 are required for BUB-1 localisation between segregating chromosomes during early anaphase I. We also show that CLS-2 is subject to SUMO-mediated regulation; CLS-2 precociously localises in the midbivalent when either SUMO or GEI-17 are depleted. Overall, we provide evidence for a novel, SUMO-mediated control of protein dynamics during early anaphase I in oocytes.

. Localisation of SUMO and AIR-2 in live oocytes. mCherry::SUMO and GFP::AIR-2 were followed during anaphase I using strain FGP5. Scale bar, 2 µm.     In the absence of SUMO or GEI-17, most ring domain components are not present in the midbivalent or the central-spindle. Conversely, The CLASP orthologue CLS-2 populates this region prematurely. In the absence of ULP-1, BUB-1 and SUMO failed to be completely removed from the spindle during anaphase. Table S1. Strains used in current study.

Movie 1
Movie montage of meiosis I in GFP::SUMO (green) and mCherry::H2B (magenta) expressing oocytes (FGP9). Images, which are the maximum projection of 3 z-sections, were collected every 30 seconds. Scale bar, 10 μm. First frame indicates the intensity scale for each channel and Gaussian blur applied to the movie.

Movie 2
Movie montage of meiosis I in GFP::SUMO (green) and BUB-1::mCherry (magenta) expressing oocytes (FGP26). Images, which are the maximum projection of 3 z-sections, were collected every 30 seconds. Scale bar, 10 μm. First frame indicates the intensity scale for each channel and Gaussian blur applied to the movie.

Movie 3
Movie montage of meiosis I in BUB-1::GFP (green) and mCherry::H2B (magenta) expressing oocytes (FGP51) in control (top) and smo-1(RNAi) (bottom). Images, which are the maximum projection of 2 z-sections, were collected every 30 seconds. Scale bar, 10 μm. First frame indicates the intensity scale for each channel and Gaussian blur applied to the movie.

Movie 4
Movie montage of meiosis I in GFP::ULP-1 (green) and mCherry::H2B (magenta) expressing oocytes (FGP42). Images, which are the maximum projection of 3 z-sections, were collected every 20 seconds with 2x2 binning. Scale bar, 10 μm. First frame indicates the intensity scale for each channel and Gaussian blur applied to the movie.

Movie 5
Movie montage of the first embryonic mitosis in GFP::ULP-1 (green) and mCherry::H2B (magenta) expressing oocytes (FGP42). Images, which are the maximum projection of 3 zsections, were collected every 20 seconds with 2x2 binning. Scale bar, 10 μm. First frame indicates the intensity scale for each channel and Gaussian blur applied to the movie.

Movie 6
Movie montage of meiosis I in CLS-2::GFP (green) and mCherry::H2B (magenta) expressing oocytes (JDU38) in control (top) and smo-1(RNAi) (bottom). Images, which are the maximum projection of 2 z-sections, were collected every 30 seconds. Scale bar, 10 μm. First frame indicates the intensity scale for each channel and Gaussian blur applied to the movie.

Movie 7
Movie montage of meiosis I in BUB-1::linker::AID::GFP (green) and mCherry::H2B (magenta) expressing oocytes (FGP77) from control (top) or auxin-treated (bottom) worms. Images, which are the maximum projection of 3 z-sections, were collected every 20 seconds. Scale bar, 10 μm. First frame indicates the intensity scale for each channel and Gaussian blur applied to the movie.

Movie 8
Movie montage of meiosis I in CLS-2::linker::AID::GFP (green) and mCherry::H2B (magenta) expressing oocytes (FGP103) from control (top) or auxin-treated (bottom) worms. Images, which are the maximum projection of 3 z-sections, were collected every 20 seconds. Scale bar, 10 μm. First frame indicates the intensity scale for each channel and Gaussian blur applied to the movie.