Description

Topoisomerase II is a homodimeric, ATP-dependent enzyme that is conserved across species (Dong and Berger 2007; Nitiss 2009; Kim, Lee, and Koo 2000; Pommier et al. 2016; Vos et al. 2011). Topoisomerase II (Topo II) is expressed in all eukaryotic cells and is essential for many cellular processes including replication, transcription, chromosome structure, and chromosome segregation during mitosis (Nitiss 2009). In meiosis, Topo II is required for proper homologous chromosome segregation at the first meiotic division (Jaramillo-Lambert et al. 2016; Rose, Thomas, and Holm 1990; Marchetti et al. 2001; Hughes and Hawley 2014). In C. elegans, the complete absence of the Topo II homolog, TOP-2, results in sterility, while a loss-of-function mutation, top-2(it7), exhibits a sex-specific chromosome segregation defect at anaphase I of meiosis in spermatogenesis. This chromosome segregation defect is not observed during oogenesis (Jaramillo-Lambert et al. 2016). Previously, we generated a TOP-2::3XFLAG strain to visualize TOP-2 localization in dissected gonads (Jaramillo-Lambert et al. 2016). Probing for anti-FLAG on dissected gonads of the TOP-2::3XFLAG strain, we observed nucleoplasmic TOP-2 localization in the mitotic proliferative zone, which then becomes associated with chromosomes during the transition zone, pachytene, and diplotene regions of the germ line. During late meiotic prophase I (diakinesis), TOP-2 completely dissociates from the chromosomes leaving a nucleoplasmic pool in the oocytes. In order to visualize the dynamic localization of TOP-2 during meiosis in C. elegans using live imaging, we created a TOP-2::GFP tagged strain via CRISPR/Cas9 genome editing at the endogenous top-2 locus. The endogenous C-terminal TOP-2::GFP is expressed in nuclei throughout the worm soma, germ line, and in developing embryos (Figure 1A-F). In the germ line, TOP-2 is expressed from the mitotic zone at the distal tip to the most proximal oocyte, which is preparing for fertilization (Figure 1A). During pachytene, TOP-2 is localized to the chromosomes (Figure 1D) similar to that observed in the anti-FLAG staining of TOP-2::3XFLAG germ lines. Interestingly, we observed a single TOP-2::GFP focus in pachytene nuclei (Figure 1D, white arrowheads). These foci are not observed with the anti-FLAG antibody staining in the TOP-2::3XFLAG germ lines. In contrast to the strictly nucleoplasmic pool of TOP-2::3XFLAG during late meiotic prophase I (diakinesis) of oocytes, we observe robust TOP-2 localization on the chromosomes of all diakinesis oocytes in addition to nucleoplasmic TOP-2::GFP (Figure 1E).

To determine whether the addition of the GFP protein tag impacts the function of TOP-2 we examined the effects on embryonic viability and brood size in the top-2::gfp worm strain. Overall brood size of the top-2::gfp worm strain was slightly reduced in top-2::gfp worms [243 (wild type, N2) vs. 194 (top-2::gfp), p=0.0001, Figure 1G]. The slight reduction in brood size in top-2::gfp is consistent with the top-2::3Xflag (Avg brood=141.5) strain we generated in a previous study (Jaramillo-Lambert et al. 2016). The reduction in brood size could either be due to minor disruptions in TOP-2 function in mitotically dividing cells within the proliferative zone of the germ line or due to TOP-2 defects in sperm production. As we aim to use the TOP-2::GFP line to study the role of TOP-2 in male meiosis and spermatogenesis, future experiments will help resolve the two possibilities. However, embryonic viability of top-2::gfp worms is not significantly different from wild-type embryonic viability [99.1% (wild type) vs. 98.9% (top-2::gfp), p= 0.79, Figure 1H]. In addition, we monitored the TOP-2::GFP strain for a high incidence of males phenotype, which is the consequence of X-chromosome nondisjunction. X-chromosome nondisjunction in wild-type worms is rare with less than 0.3% male progeny in self-fertilizing hermaphrodites (Hodgkin, Horvitz, and Brenner 1979; Lui and Colaiácovo 2013). The percent of males produced in top-2::gfp was not significantly different than wild-type worms [0% (wild type) vs. 0.6% (top-2::gfp), p=0.06]. These results indicate that the addition of the GFP protein tag to TOP-2 is not disrupting the overall meiotic functions of this important enzyme. We conclude that the endogenous C-terminally tagged TOP-2::GFP creates a functional TOP-2 protein that is suitable for endogenous protein localization studies.