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. 2010 Sep;12(9):894-901.
doi: 10.1038/ncb2093. Epub 2010 Aug 22.

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

Julien Dumont  1 , Karen Oegema, Arshad Desai
Affiliations

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

Julien Dumont et al. Nat Cell Biol. 2010 Sep.

Abstract

Although assembly of acentrosomal meiotic spindles has been extensively studied, 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 cohesion. 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.

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Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Figures

Figure 1
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 of cup-shaped meiotic kinetochores. Bottom. Localization of 6 kinetochore components and the chromokinesin KLP-19 on individual meiosis I bivalent chromosomes (see also Fig. S1). Schematic summarizes assembly dependencies for the meiotic kinetochore. Scale bar, 1 μm. (b) Top row: Schematic of chromosome segregation during meiosis I and II. (PB1: First Polar Body; PB2: Second Polar Body; PN: Pronucleus). 3 Bottom rows: stills from movies of fertilized oocytes expressing GFP-histone-H2b. Time relative to Metaphase I is in the lower right corner of each panel. White arrows indicate lagging chromosomes during anaphase I and II in the KNL-1 depletion. Quantification of lagging chromosomes during anaphase I (MI) and anaphase II (MII) is on the right. Scale bar, 5 μm. (c) KLP-19 depletion causes increased chromosome dispersal in late anaphase that is correlated with spindle instability at this stage. (d) Area occupied by the chromosomes (orange) was measured at a similar time after anaphase I onset (Control, 11.4±1.2 min; KLP-19-depleted, 11.9±0.6 min); spindle instability was observed in 8/8 KLP-19-depleted fixed oocytes at this specific cell cycle stage. Scale bars, 5 μm.
Figure 2
Figure 2. KNL-1 is required to orient chromosomes on the acentrosomal meiotic spindle prior to anaphase onset
(a) GFP-Aurora BAIR-2 and mCherry-histone-H2b dynamics in control embryos. Time is in seconds relative to anaphase I onset. Scale bar, 5 μm. (b) Top: Schematics of Aurora BAIR-2 signal on an individual chromosome and measurement of chromosome orientation angle using this signal; the spindle axis is defined by anaphase chromosome separation. Bottom: Stills from movies of the indicated conditions; quantification of individual chromosome angles relative to the axis of the meiosis I spindle, measured 20 sec before anaphase onset (n=12 embryos per condition), is presented on the right. Scale bars, 5 μm.
Figure 3
Figure 3. Anaphase chromosome separation on acentrosomal meiotic spindles occurs by a kinetochore-independent mechanism
(a) Anaphase I in control, KNL-1-depletion and KLP-19 depletion. Scale bar, 5 μm. (b) Kymographs initiated at anaphase I onset; in the KNL-1 depletion the signal in the middle is a lagging chromosome. The time interval between consecutive strips is 20 sec. (c) Graph plotting average chromosome mass separation versus time, aligned with respect to the onset of anaphase I. The average separation speed is ~0.5 μm/min for all 3 conditions. Error bars represent the standard deviation. (d) & (e) & (f) Top: Kymographs from timelapse sequences of control embryos co-expressing mCherry-histone-H2b and GFP-α-tubulin (d) or KNL-1-GFP (e) or DHC-1-GFP (f). Bottom: Stills from timelapse sequences of the three strains aligned relative to anaphase onset. Normalized fluorescence intensity along a 1-pixel-wide linescan (dashed line) is plotted on the right; position 0 corresponds to the left edge of the linescan. Scale bars, 1 μm.
Figure 4
Figure 4. Proteins localized to a ring-shaped domain between the kinetochores form linker structures during anaphase
(a) End-on views illustrating the localization of the indicated components to a ring-shaped domain between the two kinetochores; see schematic for summary of the layered composition. See also Fig. S5b. (b) Analysis of the targeting dependencies of the ring protein network and summary of the results. (c) End-on views illustrating that the ring 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-labeling experiment (DNA, Tubulin, BUB-1, HCP-1); the white arrow shows the persistence of a BUB-1 ring after anaphase onset. (e) BUB-1 and HCP-1 staining during early anaphase I in KNL-1-depleted embryos. Scale bars, 1 μm.
Figure 5
Figure 5. Ring domain proteins contribute to both pre-anaphase spindle assembly and anaphase separation
(a) Stills from movies of control, BUB-1-depleted, HCP-1/2-depleted or ClaspCLS-2-depleted fertilized oocytes expressing GFP-α-tubulin and mCherry-histone-H2b. Time, relative to anaphase I onset, is in the lower right corner of each panel. Scale bar, 5 μm. (b) DNA (blue), tubulin (green) and MKLP1ZEN-4 (red) labeling in a control meiotic embryo. Scale bar, 1 μm. (c) (Top) Summary of the composition of the cup-like kinetochores and of the ring domain located between the kinetochores of meiotic chromosomes. (Bottom) A model for chromosome orientation and segregation on acentrosomal meiotic spindles. See text for further details.
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