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First published online 24 April 2007
doi: 10.1242/jcs.03447

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Cortical centralspindlin and G have parallel roles in furrow initiation in early C. elegans embryos

¹ Laboratory of Genetics, University of Wisconsin – Madison, Madison, WI 53706, USA
² Laboratory of Molecular Biology, University of Wisconsin – Madison, Madison, WI 53706, USA
³ Department of Anatomy, University of Wisconsin – Madison, Madison, WI 53706, USA

View larger version (132K): [in this window] [in a new window]   Fig. 1. Nomarski images of C. elegans embryos during cytokinesis showing furrow ingression or lack of furrowing. Furrowing fails in embryos mutant for zen-4 and depleted of PAR-2 or GPR-1/2 but not in embryos mutant for spd-1 and depleted of PAR-2 or GPR-1/2. Bar, 10 µm. We selected frames in which the furrow has reached the midzone or similar timepoints in embryos that failed to furrow.

View larger version (28K): [in this window] [in a new window]   Fig. 2. Spindle length determination at the initiation of furrow ingression. (A) Time-lapse images of alternating mid- (top row) and cortical- (bottom row) planes of wild-type embryos expressing β-tubulin::GFP. Numbers represent time in seconds after the initiation of metaphase to anaphase transition. Bar, 10 µm. (B) Length of spindle at time of furrow ingression or 150 seconds after the metaphase to anaphase transition for embryos that fail to furrow (see text). Red bars represent embryos that fail to furrow. Blue bars are embryos that furrowed. Each bar represents the average length of the spindle in microns from five embryos and the error bars represent s.d. The bars are arranged by increasing spindle length.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Microtubule distribution at the cortex in live embryos. (A) Normalized number of microtubule ends along the length of the embryo at the time of furrow initiation. The number of microtubule ends in a 5 micron-wide window surrounding each position was divided by the mean of all of the values for that embryo in order to compare different embryos. Average of 10 embryos. 0 is the position where the furrow begins ingression, anterior is left (negative values) and posterior is right (positive values). There are valleys and peaks throughout the length of the embryo and yet the equatorial cortex does not align with the major minimum between the poles. Analysis of individual embryos indicates that the equatorial cortex sometimes aligns with a local maximum of microtubules and rarely aligns with the global minimum (see supplementary material Fig. S4). Similar results were obtained for all timepoints between the onset of anaphase and furrow initiation (see supplementary material Fig. S5). The steep decrease in microtubule ends is a consequence of the way in which microtubule ends are counted. The measurement windows have approximately the same lateral extent in the central region of the embryo, which is roughly cylindrical, and so the counts in this region are equivalent to microtubule density, but the area of cytoplasm fall-off in the poles, which gives rise to a reduction of microtubule end counts in these regions. See supplementary material Fig. S6 for analysis of this data using different window sizes. (B) Number of microtubule ends at the equatorial cortex before and after furrow initiation in four different wild-type embryos. Time 0 is when the furrow first appears. Each frame is 10 seconds apart. There is no consistent increase or decrease in the number of microtubule ends in the equatorial cortex at the time of furrow initiation or 20 seconds earlier when the contractile ring is expected to form. (C) Average relative number of microtubule ends around the equatorial cortex at the time of furrow initiation for various mutant combinations that can furrow. Each line is an average of three embryos. 0 is the position where the furrow begins ingression, anterior is left (negative values) and posterior is right (positive values). There is no consistent increase or decrease of microtubule ends at the equatorial cortex in embryos that furrow.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Model for furrow initiation in the C. elegans one-cell embryo. We propose that the spindle midzone, centralspindlin on the cortex and G act in parallel to initiate furrowing. Although much of the mechanism is still poorly understood, it might act through microtubule stability and localized activation of Rho. See text for more details.

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