Su KC , Bement WM , Petronczki M , von Dassow G .

R01 GM052932 NIGMS NIH HHS , GM52932 NIGMS NIH HHS , Cancer Research UK

	FIGURE 1:. Urchin Ect2 recruits to the central spindle and the astral microtubule ladder. (A) Schematic of urchin vs. human Ect2, showing positions of tags and derivatives used. (B) Wild-type 3xGFP Ect2 (gray) in 32-cell sand dollar coexpressed with mCh-H2B (red); single optical sections (Supplemental Video S1). (B′) Two-times magnification of the indicated cell immediately before, early in, and mid cleavage. (C) 3xGFP Ect2 GEF4A (gold) in 16-cell sand dollar embryo coexpressed with 2xmCh EMTB (cyan; Supplemental Video S2). (C′) Two-times magnification of the middle cell during the period when the astral ladder develops between central spindle and equatorial cortex.
	FIGURE 2:. Wild-type Ect2 induces hypercontractility during cytokinesis. (A) Sand dollar embryo expressing 3xGFP SpEct2 WT(white) and mCh-H2B (red), single section. Cells 1–4 exhibit various behaviors, likely due to variation in the amount of Ect2 mRNA inherited after injection: cell 1 cleaves normally, and cells 2–4 are hyperactive. Open arrowheads point out instances of cortical Ect2; solid arrowheads follow the midzone in cell 2 as it slips when the cell contracts. See Supplemental Video S4. (B) Eight-cell sand dollar embryo coexpressing eGFP-rGBD and wt SpEct2, projection of 15 1-μm sections; these cells exhibit broad furrow zones and ectopic Rho activation. Times are minutes:seconds after filming began.
	FIGURE 3:. Too much Ect2-G4A inhibits furrowing. (A, B), Projections of 16 1-μm (A) or 13 2-μm (B) sections of sand dollar embryos expressing sufficient 3xGFP Ect2 GEF4A to cause cytokinesis failure. Embryos failed cytokinesis once before imaging began. (A) t = 01:30 and (B) t = 44:40 end-on views through the central spindle, where Ect2 accumulates before moving to astral microtubules, indicated by open arrowheads in A. See Supplemental Video S5. Solid arrowheads point out cortical Ect2 in the ingressing furrow. (C) Sand dollar embryo expressing 3xGFP Ect2 GEF4A (gold) and 2xmCh EMTB (blue), which has undergone multiple cycles of cytokinesis failure (18 1-μm sections). As the cell progresses through anaphase, Ect2 relocates from central spindle to overlapping astral microtubule regions, which remain after nuclear reassembly. Times are minutes:seconds after filming began.
	FIGURE 4:. Ect2 and centralspindlin climb the astral ladder in starfish. (A) One of eight cells in a starfish embryo expressing wt 3xGFP SpEct2 (gold) and 2xmCh EMTB (cyan; Supplemental Video S6). (A′, A″) Two-times magnifications of the region covering central spindle and cell equator before and midway through cleavage, with Ect2 (left), merge (middle), and microtubules (right). Arrowheads in A′ indicate astral microtubules decorated by Ect2. Arrowheads in A″ indicate Ect2-decorated microtubule bundles—the ladder—that cross the equator parallel to the spindle. (B) Kymograph from same sequence as A; times of A′ and A″ shown by vertical lines. Note the diagonal spread of Ect2 from central spindle to cell surface and that the central spindle itself grows dimmer over time as the ladder grows brighter. (C) One of 16 cells in a starfish embryo expressing wt 3xGFP SpCyk4, which appears on the central spindle and faintly on astral rays throughout the cell in anaphase (first frame) and then clears from the spindle poles outward and accumulates on astral microtubules beneath the furrow (Supplemental Video S7).
	FIGURE 5:. Ect2 shapes the zone of active Rho. (A) Comparison of sand dollar embryos at the 16- to 32-cell division, expressing GFP-rGBD alone or with wt Ect2; projections of 15 (control) or 20 (Ect2) 1-μm sections (Supplemental Video S8). The dose of Ect2 used here was low enough that no overt defects were evident, but the embryo displays a broadened, brightened Rho zone. (B) Single surface section of 32-cell sand dollar embryo expressing GFP-rGBD and a dose of SpEct2 GEF4A at which no defects were apparent. False coloring was applied to highlight faint active Rho zones, which are not detectable in a projection of 11 1-μm sections displayed in the inset. (C) Representative projections of single sand dollar cells at equivalent cell sizes at onset of furrowing, injected with GFP-rGBD alone, or with low dose of wt Ect2 and GEF1A or GEF4A mutants. (D) Plot of cell size vs. width of Rho zone from images as in C. (E) Projection of all 28 (top) or four medial (lower) 0.8-μm sections of a dividing four-cell sand dollar embryo expressing eGFP-rGBD. Top right cell was also injected with mRNA encoding hyperactive SpEct2-CT and displays strong Rho activity over the entire surface.
	FIGURE 6:. Sand dollar embryo expressing 3xGFP Ect2 GEF4A (gold) and 2xCh EMTB (blue) and treated with 20 μM trichostatin A at time 00:00 (minutes:seconds); projection of four 1-μm sections. Inset, 2× magnification of region covering the lowermost cell, Ect2 only.
	FIGURE 7:. Ect2 and centralspindlin are associated with secondary furrows in toroidal cells. Starfish zygote (A) and one of four cells (B) expressing wt 3xGFP Ect2 (gold) and 2xmCh EMTB (cyan) and perforated with a 50-μm glass bead (Supplemental Video S9). Top, first division after perforation; bottom, next division of resulting binucleate, U-shaped cell. Inset in A, 2× magnification of the secondary furrow region (boxed); insets 1 and 2 in B, primary and secondary furrows. (C) One of two cells in a starfish embryo expressing wt 3xGFP Cyk4 and perforated as in A and B (Supplemental Video S10); inset, secondary furrow region. Open arrowheads mark primary (spindle-crossing) furrows; solid arrowheads mark secondary furrows that pass between two asters.
	FIGURE 8:. Centralspindlin recruits between paired asters in anucleate cytoplasts. Both panels show anucleate cytoplasts the first time they try to divide with two asters. (A) Medial view, projection of six sections; diagram depicts the operation. (A′) First cleavage of nucleate half of zygote in A (pb, polar bodies). (B) Single supramedial section; insets, 2× magnifications of boxed region; open arrows indicate astral microtubules decorated with Cyk4. See Supplemental Video S11.

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