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First published online 15 January 2008
doi: 10.1242/jcs.022897

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Cytokinetic furrowing in toroidal, binucleate and anucleate cells in C. elegans embryos

Center for Cell Dynamics, Friday Harbor Laboratories, 620 University Road, Friday Harbor, WA 98250, USA and Department of Biology, University of Washington, Seattle, WA 98195, USA

View larger version (56K): [in this window] [in a new window]   Fig. 1. Cytokinesis in toroidal C. elegans embryos. (A) Complete perforation interposes a cylinder of cell cortex between the mitotic apparatus and what would have been the equatorial cortex; no latitudinal connection exists between the cortex surrounding the mitotic apparatus and the cortex on the opposite side of the probe. (B) Frames from a DIC film of perforation and subsequent cytokinesis (supplementary material Movie 1). Frame 1 shows the dechorionated zygote at metaphase, still in its vitelline envelope. Anterior is to the left in all figures. Frame 2 is immediately following perforation with a probe 5 µm in diameter. Arrowheads indicate spindle poles; ss, spindle side, fs, far side from the perforation. A furrow forms on both the far side and spindle side (frame 3); in this case, both furrows ingress deeply (frame 4), but the far-side furrow regresses (frame 5) shortly after nuclei reappear. The spindle-side furrow completes (frame 6), yielding a U-shaped, binucleate cell. Bar, 10 µm.

View larger version (16K): [in this window] [in a new window]   Fig. 2. Results from perforation of wild-type zygotes (P0) and the anterior cell (AB) of the two-cell embryo. Numbers shown include all cases, whether filmed with transmitted light or using NMY-2::GFP, in which perforation unambiguously preceded anaphase onset. (A) Most perforated P0 cells exhibit two overt furrows, but in the vast majority only the spindle-side furrow is stable. If the probe fails to intercept the space between the spindle midzone and the equator, then typically a furrow forms around the equator, seemingly ignoring the probe. In some cases the cortex surrounding the probe participates, and may yield a U-shaped binucleate cell, or cleavage may eliminate the probe from both cells. (B) In perforated AB, overt furrowing is less frequent on the far side of the probe (although the far side always exhibits myosin recruitment; see Fig. 3). As in P0, almost all AB in which the probe lies directly between the mitotic apparatus and the far-side cortex, successfully cleave only on the spindle side, yielding a binucleate U-shaped cell.

View larger version (126K): [in this window] [in a new window]   Fig. 3. Perforated cells consistently recruit myosin II to the far-side equatorial cortex. (A) Frames from a time-lapse sequence of a perforated zygote. In frame 1 asterisks indicate the approximate position of the spindle poles, inferred from the cloud of GFP-myosin that appears on the metaphase spindle. In both unmanipulated and perforated zygotes, the first phase of cytokinetic myosin recruitment is an array of blotches (frame 2), which appear broadly on the cell surface, then rapidly resolve into an equatorial band. Brackets in frame 3 mark the extent of the equatorial myosin zone on both the spindle side and far side. Often, as in this case, the far-side myosin zone is noticeably fainter than the spindle-side zone (although see Fig. 5B). Nevertheless this weak zone coincides with a shallow, persistent furrow (this embryo was scored for two furrows in Fig. 2A). The cortex around the probe recruits myosin intensely on the side facing the spindle. (B) Frames from a time-lapse sequence of a perforated AB. Frame 1 is before nuclear envelope breakdown; myosin is present everywhere on the cortex in AB, but not P1, before anaphase. Asterisks in frame 2 mark the approximate positions of spindle poles. During anaphase, myosin disappears from the polar cortex, but remains and brightens on both the spindle-side and far-side equatorial cortex (frame 3). The cortex between the probe and the spindle also develops an intense myosin zone (frame 4; brackets indicate the approximate extent of equatorial myosin zones) and, although myosin persists on the far side cortex, in this case furrowing occurs only on the spindle side (frame 5; this embryo was scored for one furrow in Fig. 2B). Shortly after nuclei reappear (dark spots, frame 6) myosin can no longer be detected on the far-side cortex. Bar, 10 µm; ss, spindle side; fs, far side from the perforation.

View larger version (75K): [in this window] [in a new window]   Fig. 4. SPD-1 depletion fails to abolish spindle-side furrowing, and makes far-side furrowing both deeper and more persistent. (A) Time-lapse sequence of the mitotic apparatus in a SPD-1-depleted zygote. The mitotic apparatus appears completely normal in metaphase (frame 1). When spindle poles move apart in anaphase, however, no microtubules remain between the separated chromosomes (frames 2 and 3). Kinetochore fibers break down, spindle poles swell, and furrowing initiates, as in wild-type embryos (frames 4 and 5). The only trace of the midzone microtubule array is a slender bundle of astral microtubules constricted by the cytokinetic furrow (frame 6, arrowhead). (B) Perforated SPD-1-depleted zygote. Asterisks in frame 1 mark the approximate position of spindle poles. The cortex around the probe recruits myosin uniformly instead of only on the side apposed to the spindle (frames 2-4). Furrows initiate both on the far side and spindle side of the perforation (frame 3, arrowheads). Both furrows ingress to completion (frames 4-6). (C) Perforated SPD-1-depleted AB. Asterisks mark approximate positions of spindle poles. Myosin recruitment takes place on the spindle side equatorial cortex, along the spindle side of the probe, and on the far side equatorial cortex (bracket in frame 2). Both far side and spindle side develop ingressions (frame 3; arrowhead in frames 3-5 marks the far-side furrow); at the time the spindle-side furrow completes (frame 4) the far side maintains a myosin-enriched furrow which ingresses deeply (frame 5), but ultimately regresses (frame 6). Bar, 10 µm; ss, spindle side; fs, far side from the perforation.

View larger version (86K): [in this window] [in a new window]   Fig. 5. Rappaport furrows in C. elegans. (A) A sequence of DIC images in which the spindle-side furrow failed to abscise (frame 3), yielding a toroidal binucleate cell. Since no cleavage scar exists to orient the posterior spindle, at second cleavage both spindles develop in parallel. Furrows form both across each spindle (arrowheads) and between the two pairs of spindle poles (arrowheads with `r' for Rappaport furrow). Both the spindle-crossing and Rappaport furrows may ingress completely (frame 5) and remain stable, but Rappaport furrows are less likely to do so (one has regressed in frame 6; the other is stable). (B) A wild-type, perforated GFP-myosin-expressing zygote that exhibits furrowing between two unconnected spindle poles. Frames 1-3 show that this cell exhibited myosin recruitment to both the spindle-side and far-side cortex, and that as usual only the spindle-side furrow completed, yielding a U-shaped binucleate cell (frame 4). In this case the posterior spindle failed to align with the AP axis; asterisks in frame 5 indicate spindle poles. During cytokinesis three furrows initiate: around the anterior arm of the U, around the posterior arm, and between the two spindles (arrowheads in frame 7). The middle furrow represents an independent induction since the posterior arm of the U exhibits myosin recruitment to the probe cortex (arrowhead in frame 8). (C) A perforated, PAR-2-depleted zygote. Frame 1 shows that a furrow closed on the spindle side, yielding a binucleate U-shaped cell. Asterisks in frame 3 indicate the spindle poles. Furrowing begins across each spindle (frame 4), but myosin is also recruited between them, eventually resulting in a shallow but persistent furrow (frames 5 and 6). Bars, 10 µm; ss, spindle side; fs, far side from the perforation; r, Rappaport furrow.

View larger version (157K): [in this window] [in a new window]   Fig. 6. Binucleate cells cleave from one to four. (A) A sequence of DIC images in which AB and P0 were fused in interphase. We fired several pulses from the UV laser at the cell-cell interface (`x' in frame 1). The hole rapidly widens as cytoplasm flows through; in frame 2 the arrowhead marks the edge of a curtain of withdrawing membrane. In this case, the nuclei remained separate during interphase (frame 3) and two independent spindles, oriented in parallel, developed at metaphase (frame 4). Furrows ingressed between each pair of spindle poles regardless of whether they shared a spindle (arrowheads in frame 5 indicate furrows; the fourth furrow, on the anterior, appeared later; `r' for Rappaport indicates furrows that apparently bisect unconnected spindle poles). (B) Identical case to (A) but conducted in an embryo expressing both GFP-tubulin and GFP-myosin (supplementary material Movie 2). In frame 1 `x' indicates the fusion site; arrowheads in frame 2 indicate margins of the spreading hole. Nuclear envelope breakdown and spindle assembly occur sooner in the anterior nucleus (frame 3); two mitotic spindles form in parallel with no apparent connection between them (frames 4 and 5). Furrows initiate between each pair of spindle poles (arrowheads in frame 6). All furrows ingress deeply, meet near the center, and complete (frames 7-9), creating a four-cell embryo with an abnormal arrangement of cells. Bars, 10 µm.

View larger version (84K): [in this window] [in a new window]   Fig. 7. Anucleate cells attempt cytokinesis. In each case one spindle pole was cut away from the rest of the mitotic apparatus (dashed line). (A) Frames from a DIC sequence in which the anterior spindle pole was severed. Arrowheads in frames 1 and 2 indicate the spindle poles immediately before and after severing. Cytokinesis partitions both daughter nuclei into the posterior cell (dots in frame 3); the anterior cell contains no visible nucleus, only a centrosome. After duplicating, centrosomes in the anucleate AB adopt opposite positions in the cell and a furrow develops between them (frame 4; arrowheads show centrosome position), ingresses deeply (frame 5), but eventually regresses (frame 6). P1 divides normally (frame 6). The anucleate AB attempts division repeatedly: frame 7 shows the second attempt (other furrows exist in other focal planes, as this cell attempts to divide into four) and frame 9 shows the third (at which point at least four, and probably eight, cytoplasmic domains are partitioned transiently by furrows). (B) The same experiment conducted in an embryo expressing both GFP-gamma tubulin and GFP-histone. Frames 1 and 2 show the metaphase spindle immediately before and after severing. Arrowhead marks the severed centrosome; although bleached by the laser, it recovers fluorescence rapidly (frame 3). After furrowing is completed, no chromatin is detected in AB. As in (A), the duplicated centrosomes adopt opposite positions (frame 5), and a furrow develops (arrowhead in frame 6). P1 enters metaphase as the furrow regresses in AB (frame 7), then divides normally, albeit late relative to AB (frame 8). In frame 9 AB attempts another division (see supplementary material Movie 3, which shows a similar case). (C) An embryo in which the posterior spindle pole was cut away at metaphase. Duplicated centrosomes appear as tiny dots (frame 4) which swell as the cell enters mitosis (frame 5), and, strikingly, adopt an alignment within the cell corresponding to the normal alignment of the spindle in P1; no chromatin is present between the centrosomes (frame 6). A deeply-ingressing furrow develops between the centrosomes in the anucleate P1 (frames 7-9; arrowheads in frame 7). This furrow apparently completed since it persisted at least 22 minutes. Bars, 10 µm.