Fig 1.
Depletion of KLP-15/16 results in disorganized oocyte spindles.
(A) Schematic of KLP-15/16 with the proline-rich tail in orange, coiled-coil domain in white, and the motor domain in blue. The dotted line represents where klp-15(RNAi) targets, and the dashed line represents where klp-16(RNAi) targets. The underlined region labeled “Ab” is the region of the protein our antibody was made against. (B) Movie stills from control and klp-15/16(RNAi) oocytes expressing GFP::tubulin and mCherry::histone. In the control, after NEBD, diffuse tubulin can be seen inside the nucleus as was recently reported [20]. Then microtubules are nucleated and bundled to form a cage-like structure before reorganizing into a multipolar spindle and achieving bipolarity (in 5/5 control movies examined, spindles achieved bipolarity prior to anaphase onset). Following klp-15/16(RNAi), microtubules form a transient cage that then forms a disorganized array that fails to achieve bipolarity and collapses into a microtubule ball (in 6/6 klp-15/16 (RNAi) movies examined, spindles entered anaphase without achieving bipolarity); although strong cage bundles are not evident in this particular example, fixed imaging has confirmed that they are able to form. (C, D, and E) Meiotic (C and D) and mitotic (E) spindles stained for DNA (blue), tubulin (green) and ASPM-1 (red). (C) In the control, ASPM-1 marks spindle poles at the multipolar and bipolar stages. In klp-15/16(RNAi) oocytes, the microtubule cage forms, but then aberrant structures form with diffuse ASPM-1 staining. (D) Line scans of metaphase spindles in control and klp-15/16(RNAi) oocytes. 6 z-slice sum projections of spindles were used for the analysis and the average fluorescence intensities are graphed (solid line) with the SEM (shaded area). The y-axes of the graphs are the same for the control and experimental conditions for a given channel. (E) Mitotic spindles are normal in klp-15/16(RNAi) embryos (12/12 klp-15/16(RNAi) spindles analyzed were bipolar and indistinguishable from control spindles). Bars = (B) 5 μm; (C and D) 2.5 μm; (E) 10 μm.
Fig 2.
KLP-15/16 localize to spindle microtubules during oocyte meiosis.
(A) Western blot from control, klp-15 or klp-16(RNAi) worms probed with the KLP-16 antibody or tubulin as a loading control. (B and C) DNA (blue), tubulin (green) and KLP-15/16 (red). (B) Wild-type meiotic spindles at all stages of spindle assembly; KLP-15/16 begin to accumulate on microtubules at the multipolar stage and remain associated through anaphase (quantification in Materials and Methods). (C) The KLP-15/16 signal is lost following klp-15/16(RNAi), though staining of klp-15(ok1958) and klp-16(wig1) is not different from wild-type spindles (quantification in Materials and Methods). (D and E) Live imaging of worms expressing KLP-16::GFP and mCherry::histone shows that KLP-16 localizes to microtubule bundles at the cage stage (which was not apparent in the KLP-16 antibody staining) and remains associated with the spindle (D). The multipolar image is a partial projection of the entire structure. Stages of spindle assembly were discerned by spindle morphology, chromosome organization, and position in the germline. (E) KLP-16 is also present on mitotic spindle microtubules and centrosomes at the one-cell stage (in 5/5 embryos analyzed). Bars = (B, C, and D) 2.5 μm; (E) 10 μm.
Fig 3.
Microtubules bundle and chromosomes segregate in anaphase in klp-15/16(RNAi) oocytes.
(A) DNA (blue), tubulin (green), AIR-2 (not in merge) and SEP-1 (red in merge), showing anaphase progression in control and klp-15/16(RNAi) oocytes. At metaphase, AIR-2 is in the midbivalent ring and SEP-1 displays a staining pattern characteristic of outer kinetochore proteins in C. elegans oocytes (cupping the bivalents and also forming filamentous linear structures within the spindle [61]). In early anaphase, SEP-1 co-localizes with AIR-2 in the rings, and in late anaphase, SEP-1 is gone and AIR-2 is relocalized onto the microtubules. In klp-15/16(RNAi) oocytes, microtubules are disorganized in metaphase and early anaphase, but in late anaphase microtubules are bundled and chromosomes segregate into distinct masses; note that SEP-1 also localizes to the cell cortex in late anaphase. n represents the number of spindles observed for each condition. (B and C) Anaphase spindles with DNA (blue), tubulin (green), and ASPM-1 (B) or KLP-18 (C) (red). (B) Line scans of anaphase spindles in control and klp-15/16(RNAi) oocytes. The top row for each condition is a max projection of the entire spindle and the image used for the line scan is a 10 z-slice sum projection. ASPM-1 is enriched at the poles in control spindles (21/24 of spindles analyzed had ASPM-1 enriched at two poles; 88%), but is diffuse along spindle microtubules in anaphase of klp-15/16(RNAi) oocytes (only 9/26 spindles could be classified as having any type of ASPM-1 enrichment at pole-like foci; 35%); in both cases ASPM-1 also displays cortical localization. (C) KLP-18 is enriched at spindle poles in the control (11/12 spindles analyzed; 92%), but is localized throughout the spindles in klp-15/16(RNAi) oocytes (6/6 spindles had diffuse KLP-18 localization). (D) Percentage of aneuploid MII embryos from control and klp-15/16(RNAi) worms. Chromosomes were counted in images of MII embryos, and embryos were counted as aneuploid if the number of chromosomes did not equal 6. Bars = 2.5 μm.
Fig 4.
SPD-1 and centralspindlin localize to anaphase spindle microtubules.
(A and B) DNA (blue), tubulin (green) and ZEN-4 (top) or SPD-1 (bottom) (red) in control (A) or klp-15/16(RNAi) (B) oocytes. ZEN-4 does not localize to metaphase spindles in either case. In anaphase, ZEN-4 localizes to a distinct region in the midzone of control spindles (A, top zoom), but is sometimes not seen on spindles with a short chromosome segregation distance. Following klp-15/16(RNAi), ZEN-4 localizes to the microtubule bundles but is not concentrated to as distinct a band as in the control (B, top zoom). In both control and klp-15/16(RNAi) oocytes, SPD-1 does not localize to metaphase spindles. In control oocytes, SPD-1 localizes to the microtubules between the chromosomes in early anaphase and then becomes concentrated in a band on the microtubule bundles in the center of the spindle (A, bottom zoom). Following klp-15/16(RNAi), SPD-1 localizes to microtubule bundles between segregating chromosomes, even when bundles are not all oriented along the same axis (B, bottom zoom) (quantification in Materials and Methods). (C) DNA (blue), tubulin (green), ZEN-4 (red) and SPD-1 (green in final merge). In both control and klp-15/16(RNAi) oocytes, ZEN-4 and SPD-1 have similar but distinct localization patterns. (D) Movie stills of oocytes expressing mCherry::histone and SPD-1::GFP in both control and klp-15/16(RNAi). In control spindles, SPD-1 localizes between chromosomes as they begin to segregate and continues to accumulate as anaphase progresses, consistent with previous studies [19, 20]. Following klp-15/16(RNAi), SPD-1 begins to load all over the microtubules, and as SPD-1 accumulates, long bundles begin to form that then orient along the same axis and the chromosomes segregate (4/4 movies analyzed). White dashed lines = cell cortex. Bars = 2.5 μm.
Fig 5.
SPD-1 and KLP-18 are required for KLP-15/16-independent spindle reorganization during anaphase.
(A and D) DNA (blue), tubulin (green), AIR-2 (not in merge) and SEP-1 (red in merge); all spindles shown are mid/late anaphase, with SEP-1 gone and AIR-2 relocalized to the microtubules. (A) Singly depleting/inhibiting klp-15/16, spd-1, or zen-4 or doubly depleting/inhibiting klp-15/16;zen-4 and spd-1;zen-4 all resulted in anaphase spindles that were able to bundle microtubules and segregate chromosomes, while double depletion of klp-15/16;spd-1 abolished microtubule bundling and chromosome segregation. (B) Quantification of the experiment shown in (A). The simple matching coefficient for microtubule bundling and chromosome segregation = 0.82 (n = 251); in other words, 82% of the spindles either showed chromosome segregation when microtubules were bundled or did not show chromosome segregation when microtubules were not bundled. (C) Box plots showing anaphase microtubule length measurements; for a given image, the most prominent and longest microtubule bundle in the spindle was measured. Box represents first quartile, median, and third quartile. Lines extend to data points within 1.5 interquartile range. Asterisks (***) represent significant difference (p < 0.001, two tailed t-test) compared to the other three conditions; (*) represents significant difference (p < 0.05, two tailed t-test) compared to control conditions. (D) Anaphase spindle reorganization and chromosome segregation are not observed in klp-18(tm2841) following either control or klp-15/16 RNAi; in both conditions microtubules are disorganized and segregation fails, suggesting that KLP-18 could potentially mediate the anaphase spindle reorganization observed in KLP-15/16-depleted oocytes. n represents the number of spindles observed for each condition. (E) DNA (blue), tubulin (green), AIR-2 (not in merge) and SPD-1 (red in merge); SPD-1 localizes to spindle microtubule bundles in klp-18(tm2841) and klp-18(tm2841);klp-15/16 (RNAi) oocytes. n represents the number of spindles observed for each condition. Bars = 2.5 μm.
Fig 6.
Consequences of the anaphase defects observed in klp-15/16(RNAi) oocytes.
(A and B) Analysis of polar bodies and maternal pronuclei was done using live worms expressing GFP::tubulin, GFP::histone. (A) Quantification of the number of polar bodies per embryo for each condition listed. (B) Quantification of the number of maternal pronuclei per embryo for each condition listed. For A and B, embryos were only scored if the paternal pronucleus was decondensed to ensure that the meiotic divisions were complete. (C) Example mitotic embryos showing DNA (blue) and tubulin (green) to show some of the phenotypes observed in the quantification displayed in A and B. Asterisks denote polar bodies and arrowheads denote maternal pronuclei. We observe extra polar bodies and pronuclei following klp-15/16(RNAi), indicative of meiotic defects. Moreover, co-depletion of KLP-15/16 and SPD-1 often results in ejection of all maternal chromosomes into a single polar body, resulting in no maternal pronucleus. Bars = 10 μm.
Fig 7.
Anaphase spindle organization in klp-15/16(RNAi) oocytes.
(A) DNA (blue), tubulin (green in columns 1, 4, 5, 6, 7), SUMO (red in columns 4 and 5, green in columns 8 and 9), and SPD-1 (red, columns 6, 7, 8, 9); for each color combination, max projections of the full spindle are labeled “full” and the single z-slices used for the line scans are labeled “z-slice”. In control and klp-15/16(RNAi) oocytes, chromosomes can be seen segregating through channels with SUMO in the middle of the channel flanked by SPD-1 on the microtubule bundles. Line scans across the channels in single z-slice images show colocalization of microtubules and SPD-1 and anti-correlation of SUMO/microtubules and SUMO/SPD-1; these oscillations were observed in 9/11 control anaphase spindles (82%) and 12/18 klp-15/16(RNAi) anaphase spindles (67%). However, following klp-15/16(RNAi), some rings also appear to be on the periphery of the spindle (arrowheads). (B) Single z-slice images of DNA (blue), tubulin (green), SUMO (red, columns 1, 2, 4), and SPD-1 (red, column 3). In early anaphase following klp-15/16(RNAi), rings are dissociated from chromosomes and are embedded within the microtubules of the spindle, or are excluded from the spindle. In late anaphase, the rings can be within channels between segregating chromosomes or outside of a channel not associated with a segregating chromosome pair. Arrows indicate examples of lateral microtubule-chromosome associations. Within each row, the late anaphase images are different z-slices from the same spindle, chosen to highlight different features. (C) DNA (blue) and tubulin (green); single z-slice images showing additional examples of lateral microtubule-chromosome associations in klp-15/16(RNAi) oocytes. We observed clear lateral associations in 31/35 control anaphase spindles (89%) and in 22/31 klp-15/16(RNAi) anaphase spindles (71%). Bars = 2.5 μm.
Fig 8.
Interplay between microtubule-associated factors contributes to acentriolar spindle assembly and maintenance.
DNA (blue), microtubules (green), minus-end/pole marker (orange), ring complex (red), and SPD-1 (yellow). In wild-type spindles, KLP-15/16 load onto microtubules during the cage stage and remain associated through anaphase providing stability to microtubule bundles. At anaphase onset, SPD-1 loads onto the microtubules as the chromosomes begin to segregate towards the poles through microtubule channels (Anaphase A), and the ring complexes are left behind inside the channels. As anaphase progresses, the spindle microtubules elongate forcing the chromosomes further apart (Anaphase B), and the ring complexes disassemble. In klp-15/16(RNAi) oocytes, after the cage stage, microtubules form a disorganized array with ASPM-1 located throughout the structure. The array collapses into a microtubule ball around the chromosomes with ASPM-1 throughout. As anaphase begins, SPD-1 binds to the short, randomly oriented microtubules and bundles them, the ring complexes dissociate from the chromosomes, and KLP-18 sorts and aligns the bundles such that lateral microtubule-chromosome associations and channels can form. The spindle then elongates (Anaphase B) to facilitate chromosome segregation.