Levels of the ubiquitin ligase substrate adaptor MEL-26 are inversely correlated with MEI-1/katanin microtubule-severing activity during both meiosis and mitosis

Abstract

The MEI-1/MEI-2 microtubule-severing complex, katanin, is required for oocyte meiotic spindle formation and function in C. elegans, but the microtubule-severing activity must be quickly downregulated so that it does not interfere with formation of the first mitotic spindle. Post-meiotic MEI-1 inactivation is accomplished by two parallel protein degradation pathways, one of which requires MEL-26, the substrate-specific adaptor that recruits MEI-1 to a CUL-3 based ubiquitin ligase. Here we address the question of how MEL-26 mediated MEI-1 degradation is triggered only after the completion of MEI-1's meiotic function. We find that MEL-26 is present only at low levels until the completion of meiosis, after which protein levels increase substantially, likely increasing the post-meiotic degradation of MEI-1. During meiosis, MEL-26 levels are kept low by the action of another type of ubiquitin ligase, which contains CUL-2. However, we find that the low levels of meiotic MEL-26 have a subtle function, acting to moderate MEI-1 activity during meiosis. We also show that MEI-1 is the only essential target for MEL-26, and possibly for the E3 ubiquitin ligase CUL-3, but the upstream ubiquitin ligase activating enzyme RFL-1 has additional essential targets.

Fig. 1

Time course of MEL-26 accumulation in wild-type embryos. The left column shows deconvolved indirect immunofluorescence images of embryos stained with anti-MEL-26 (red) while the right column shows merged images of the same embryos stained with anti-MEL-26 (red), anti-tubulin (green) and DAPI (blue). (A, B) meiosis I. (C, D) Meiosis II, note the first polar body (arrow). (E, F) Pronuclear formation, levels of MEL-26 have increased. (G, H) Pronuclear fusion. (I, J) Anaphase of the first mitotic division. (K, L) Late two cell stage, and (M, N) four cell stage. Note that MEL-26 is enriched at the membrane between cells of multicellular embryos. (O, P) Two cell mel-26(ct61sb4); tbb-2(sb26) shows no MEL-26 staining. The mutation encoded by the allele ct61sb4 results in a truncation N-terminal to the region used to raise the antisera. tbb-2(sb26) suppresses ct61sb4 lethality, restoring normal embryo morphology. Note that the low levels of MEL-26 present at meiosis I and II (A–D) are above the background seen in (O, P).

Fig. 2

Mutations affecting MEL-26 temporal expression. Merged images are of MEL-26 (red), tubulin (green) and DAPI (blue). (A) Low levels of MEL-26 are apparent in the meiotic stage wild-type embryo (i) compared to the pronuclear fusion stage wild-type embryo (ii) in the same field. cul-2 (B) and rfl-1 (C) embryos show increased MEL-26 staining at meiosis compared to wild type. (D) emb-27 embryo arrested at meiosis I shows low levels of MEL-26. (E) zyg-11 embryos have a prolonged meiosis (as do cul-2 embryos), but levels of meiotic MEL-26 do not increase as they do in cul-2. Prolonged meiosis may result in very low levels of MEL-26 in (D) and (E). (F) The increased levels of meiotic MEL-26 characteristic of cul-2 are epistatic to the low levels seen in emb-27 in the cul-2; emb-27 double mutant. (G) CUL-2 prevents premature accumulation of MEL-26. The histogram shows the percent of embryos showing very low (white), moderate (grey) and high (black) levels of MEL-26 at the stages indicated for each genotype. Wild-type embryos show an abrupt increase in MEL-26 after meiosis. cul-2 embryos are strongly positive at meiosis I and increase slightly thereafter. An APC subunit (emb-27) mutant arrests at meiosis I and is similar to wild type at that stage, but cul-2 is epistatic to emb-27 as doubly mutant embryos are similar to cul-2. The numbers of embryos scored are as follows. Meiosis I: wild type (17), cul-2 (29), emb-27 (19), cul-2; emb-27 (25). Meiosis II: wild type (17), cul-2 (16). Prometaphase: wild type (30), cul-2 (20).

Fig. 3

Meiotic cytoplasmic MEI-1 levels are not altered in cul-2 embryos. (A–D) Embryos stained with anti-MEI. (A′–D′) Anti-tubulin staining of the same embryos. In each panel, levels in meiotic embryos are compared to the mitotic embryo present in the same field. (A, A′) Wild-type cytoplasmic MEI-1 staining is higher in the meiotic embryo than a later-stage embryo. (B, B′) cul-2 has higher levels of cytoplasmic MEI-1 at meiosis than mitosis, and levels at both divisions are higher than corresponding wild-type embryos. (C, C′) egg-3 (RNAi) embryos show similar cytoplasmic MEI-1 levels at meiosis and mitosis, indicative of premature MEI-1 degradation. (D, D′) cul-2; egg-3(RNAi) embryos have similar levels of cytoplasmic MEI-1 at meiosis and mitosis, like egg-3 alone, but cytoplasmic levels at both divisions are increased, as seen with cul-2 single mutants. Note that in all panels meiotic spindles (arrows) are present and contain MEI-1.

Fig. 4

Decreased MEI-1 degradation during meiosis leads to shorter meiotic metaphase spindles. C. elegans meiotic spindles initially maintain a constant length during metaphase I and metaphase II (Yang et al., 2003). However, just prior to anaphase they shorten and rotate from parallel to perpendicular to the cortex. The constant lengths of metaphase I and metaphase II spindles prior to shortening and their lengths at rotation were determined from time-lapse sequences of GFP: tubulin in both wild-type and mei-1(ct46) embryos in utero. Representative images of metaphase spindles (scale bar, 5 μm) and average metaphase spindle lengths are shown and tabular data are presented +/− the standard deviation. n =the number of individual spindles used to determine each average.

Fig. 5

Model of MEI-1 regulation during the meiosis to mitosis transition. Low levels of protein or activity are indicated by lighter shading. During meiosis, CUL-2 (this report) and EGG-3 (Stitzel et al., 2007) keep MEL-26 and MBK-2 activities low, respectively, allowing MEI-1 to accumulate and sever microtubules. It is not known if the interaction between CUL-2 and MEL-26 is direct, and so this interaction is shown with stippled lines. During mitosis the situation is reversed and MEI-1 is degraded, allowing formation of longer microtubules. The kinase that acts in concert with the CUL-3/MEL-26 ubiquitin ligase and the ubiquitin ligase acting with MBK-2 are unknown.