A kinetochore-independent mechanism drives anaphase chromosome separation during acentrosomal meiosis

Journal name:
Nature Cell Biology
Volume:
12,
Pages:
894–901
Year published:
DOI:
doi:10.1038/ncb2093
Received
Accepted
Published online

Although assembly of acentrosomal meiotic spindles has been extensively studied1, little is known about the segregation of chromosomes on these spindles. Here, we show in Caenorhabditis elegans oocytes that the kinetochore protein, KNL-1, directs assembly of meiotic kinetochores that orient chromosomes. However, in contrast to mitosis, chromosome separation during meiotic anaphase is kinetochore-independent. Before anaphase, meiotic kinetochores and spindle poles disassemble along with the microtubules on the poleward side of chromosomes. During anaphase, microtubules then form between the separating chromosomes. Functional analysis implicated a set of proteins that localize to a ring-shaped domain between kinetochores during pre-anaphase spindle assembly and anaphase separation. These proteins are localized by the chromosomal passenger complex, which regulates the loss of meiotic chromosome cohesion2, 3, 4. Thus, meiotic segregation in C. elegans is a two-stage process, where kinetochores orient chromosomes, but are then dispensable for their separation. We suggest that separation is controlled by a meiosis-specific chromosomal domain to coordinate cohesin removal and chromosome segregation.

At a glance

Figures

  1. Cup-shaped meiotic kinetochores are assembled by a KNL-1-dependent mechanism and are required for accurate meiotic chromosome segregation.
    Figure 1: Cup-shaped meiotic kinetochores are assembled by a KNL-1-dependent mechanism and are required for accurate meiotic chromosome segregation.

    (a) Top: schematic representation of cup-shaped meiotic kinetochores. Bottom left: localization of six kinetochore components and the chromokinesin KLP-19 on individual meiosis I bivalent chromosomes in control cells and in cells with the indicated RNAi depletions (see also Supplementary Information, Fig. S1). Bottom right: a schematic representation of the meiotic kinetochore assembly pathway. Scale bar, 1 μm. (b) Top row: schematic representation of chromosome segregation during meiosis I and II. (PB1, first polar body; PB2, second polar body; PN, pronucleus). Bottom rows: images from time-lapse microscopy of fertilized oocytes expressing GFP–H2B. Time relative to metaphase I is indicated in the lower right corner of each panel. In the KNL-1 depletion images, the white arrows indicate lagging chromosomes during anaphase I and II. Quantification of lagging chromosomes during anaphase I (meiosis I, MI) and anaphase II (meiosis II, MII) is shown on the right. Scale bar, 5 μm. (c) KLP-19 depletion causes increased chromosome dispersal in late anaphase. Area occupied by the chromosomes (orange) was measured at a similar time after anaphase I onset (Control, 11.4 ± 1.2 min and KLP-19-depleted, 11.9 ± 0.6 min). Scale bar, 5 μm. (d) KLP-19 depletion caused spindle instability in late anaphase. Spindle instability was observed in 8/8 KLP-19-depleted fixed oocytes at this specific cell cycle stage. Scale bar, 5 μm.

  2. KNL-1 is required to orient chromosomes on the acentrosomal meiotic spindle before anaphase onset.
    Figure 2: KNL-1 is required to orient chromosomes on the acentrosomal meiotic spindle before anaphase onset.

    (a) GFP–Aurora BAIR–2 and mCherry–H2B dynamics in control embryos. Time is shown relative to anaphase I onset. Scale bar, 5 μm. (b) Top: schematic of GFP–Aurora BAIR–2 localization on an individual chromosome and measurement of chromosome orientation angle using this signal; the spindle axis is defined by anaphase chromosome separation. Bottom: images from time-lapse microscopy movies of chromosomes from embryos treated with the indicated RNAi. Quantification of individual chromosome angles relative to the axis of the meiosis I spindle, measured 20 s before anaphase onset (n = 12 embryos per condition), is presented on the right. Scale bar, 5 μm.

  3. Anaphase chromosome separation on acentrosomal meiotic spindles occurs by a kinetochore-independent mechanism.
    Figure 3: Anaphase chromosome separation on acentrosomal meiotic spindles occurs by a kinetochore-independent mechanism.

    (a) Anaphase I in control, KNL-1-depleted and KLP-19-depleted cells. Scale bar, 5 μm. (b) Kymographs, initiated at anaphase I onset, showing migration of GFP–H2B. In the KNL-1-depleted cell, the signal in the middle is a lagging chromosome. The time interval between consecutive strips is 20 s. (c) Effect of KNL-1 and KLP-19 depletion on meiotic chromosome separation, from the onset of anaphase I. The average separation speed is approximately 0.5 μm min−1 for all three conditions. Error bars represent s.d. (df) Top: kymographs from time-lapse microscopy of control embryos co-expressing mCherry–H2B and GFP–α-tubulin (d), KNL-1–GFP (e) or DHC-1–GFP (f). The time interval between consecutive strips is 20 s. Bottom: time-lapse microscopy of the three strains imaged at the indicated times from the onset of anaphase. Graphs representing normalized fluorescence intensity along a 1-pixel-wide linescan (indicated by dashed line on the respective images) are plotted on the right; position 0 corresponds to the left edge of the linescan. Scale bars, 1 μm.

  4. Proteins that localize to a ring-shaped domain between the kinetochores form linker structures during anaphase.
    Figure 4: Proteins that localize to a ring-shaped domain between the kinetochores form linker structures during anaphase.

    (a) End-on views of chromosomes, illustrating the localization of the indicated components to a ring-shaped domain between the two kinetochores. Schematics represent a summary of the layered composition. See also Supplementary Information, Fig. S5b. (b) Analysis of the localization dependencies of proteins in the ring-shaped protein complex and a schematic summary of the results. (c) End-on views illustrating that the ring-shaped domain localization of HCP-1 and CLS-2 persists in KNL-1-depleted embryos. (d) Localization of HCP-1 and BUB-1 to linker structures between the separating chromosomes during anaphase of meiosis I. Panels are from a quadruple-labelling experiment (DNA, tubulin, BUB-1 and HCP-1). (e) BUB-1 and HCP-1 staining during early anaphase I in KNL-1-depleted embryos. Scale bars, 1 μm.

  5. Ring domain proteins contribute to both pre-anaphase spindle assembly and anaphase separation.
    Figure 5: Ring domain proteins contribute to both pre-anaphase spindle assembly and anaphase separation.

    (a) Time-lapse microscopy images of control, BUB-1-depleted, HCP-1/2-depleted or CLASPCLS–2-depleted fertilized oocytes expressing GFP–α-tubulin and mCherry–H2B. Time relative to anaphase I onset is indicated in the lower right corner of each panel. Scale bar, 5 μm. (b) DNA (blue), tubulin (green) and MKLP1ZEN–4 (red) labelling in a control meiotic embryo. Scale bar, 1 μm. (c) Top: summary of the composition of the cup-like kinetochores and of the ring-shaped domain located between the kinetochores of meiotic chromosomes. Bottom: a model for chromosome orientation and segregation on acentrosomal meiotic spindles.

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Affiliations

  1. Ludwig Institute for Cancer Research, Department of Cellular & Molecular Medicine, University of California, San Diego, La Jolla, CA 92093-0653, USA.

    • Julien Dumont,
    • Karen Oegema &
    • Arshad Desai

Contributions

All experimental data were generated by J.D., who also had primary responsibility for experimental design and data analysis. A.D. and K.O. contributed to experimental design and data analysis. J.D., A.D. and K.O. wrote the manuscript.

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The authors declare no competing financial interests.

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