Bement WM , Benink HA , von Dassow G .

GM52932 NIGMS NIH HHS , GM66050 NIGMS NIH HHS , P50 GM066050 NIGMS NIH HHS , R01 GM052932 NIGMS NIH HHS

	Figure 1. A zone of RhoA activity in echinoderm and amphibian cytokinesis. (A) Surface view of four-cell purple urchin embryo; projection of 18 1-μm sections. eGFP-rGBD begins to accumulate immediately before furrowing (arrowheads), brightens, and disappears once furrows are complete. (B) Surface view of eight-cell purple urchin embryo; projection of 18 1-μm sections. eGFP-rGBD accumulates before the furrow appears (arrowheads) at the site of the future furrow. (C) Sectional view through eight-cell green urchin embryo; projection of 16 1-μm sections. Before cleavage (00:00), eGFP-rGBD reveals uniform, cortical RhoA activity, which disappears (02:40) before localized activation of RhoA in the equator (arrowhead, 04:40), where the furrow will develop (06:40). (D) Surface view of X. laevis embryo; projection of 12 sections. eGFP-rGBD appears in narrow stripes (arrowheads) that presage furrow formation. See online supplemental material for Videos corresponding to B–D (Videos 1–3, respectively, available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1). Times are given in minutes:seconds after filming began. Bars, 25 μm.
	Figure 2. Characteristics of RhoA activity zones. (A) Single optical sections through a green urchin blastomere before, early, and late in furrowing showing zone in cross section. Bar, 25 μm. (B) Single optical sections through a blastomere from green urchin embryo showing eGFP-rGBD accumulation in spindle region. Chromosomes appear as a dark band (arrow, 15:12) that splits (arrows, 19:00) during anaphase. eGFP-rGBD highlights the centrosomes at all phases of mitosis (arrowhead, 26:00). Times are given in minutes:seconds after filming began. (C and D) Images of rGBD-eGFP (top), TRITC dextran (bottom), and difference images (middle) show that only the signal at the very cortex (C) and at the centrosome (D, arrowheads) is specific to eGFP-rGBD. Images in C are the mean of three successive frames and in D are the mean of 10 successive frames at 3-s intervals. (E and E′) eGFP-rGBD signal intensity measured along the cortex during division in green urchin. E shows raw data from single optical sections 2 min apart (from the cell shown in A), with time points ordered along the rainbow from red to violet. E′ shows curves obtained by fitting a weighted sum of a Gaussian and quadratic to the data in E. The Gaussian fits the furrow signal, whereas the quadratic fits the rest of the cortex. In this and five similar traces we performed, we note that the width of the fit Gaussian varies by 10% or less as the furrow ingresses despite the increasing curvature of the cortex. (F and F′) eGFP-rGBD signal intensity measured along the cortex during division in X. laevis embryo presented as in E and E' except that time points are 20 s apart. (G) Scatter plot of RhoA activity zone width versus cell diameter in urchin embryos; purple squares come from purple urchins; green squares come from green urchins. (H) Scatter plot of RhoA activity zone width versus cell size in X. laevis embryos. (I) Surface view of deconvolved series showing that eGFP-rGBD concentration (asterisk) precedes furrowing (<) in purple urchin embryos; frames are 20 s apart. (J) Z image series showing that eGFP-rGBD concentration (asterisk) precedes furrowing (<) in X. laevis embryos.
	Figure 3. RhoA zones accompany divergent forms of cytokinesis. (A) Micromere formation in green urchin embryos. Projection of 12 1-μm sections of an eight-cell green urchin embryo. Dashed outlines in first frame show spindle position and orientation. Cell at top forms a circumferential furrow above the spindle midplane; in other cells, the asymmetric position of the spindle corresponds to asymmetric furrowing, and the region of concentrated eGFP-rGBD spreads around the circumference as furrowing proceeds. (B) Polar body formation in X. laevis oocytes. Projection of 13 optical sections showing that eGFP-rGBD becomes concentrated in a circular region at the animal pole and constricts inward. Arrowheads indicate edges of zone. (C) Dual-labelled images from projection of 13 optical planes showing tubulin (red) and eGFP-rGBD (green) during polar body emission. Top row shows facing view; circular region of concentrated eGFP-rGBD closes inward around microtubules of the first meiotic spindle. Bottom three rows show Z view in which eGFP-rGBD is seen to contract inward and downward in concert with the closure of the cytokinetic array, which pinches off the forming polar body. The microscope was refocused at 05:20 to allow the eGFP-rGBD ring to be followed deeper into the cytoplasm. See online supplemental material for videos corresponding to A (Video 4) and C (Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1). All times are in minutes:seconds after filming began. Bars, 25 μm.
	Figure 4. RhoA activation in the equator does not depend on actin assembly. All times in this figure are in minutes:seconds after the addition of cytochalasin D. (A) Projection of 18 1-μm sections through a 16-cell purple urchin embryo treated with 10 μM cytochalasin D during prometaphase. As the drug takes effect, cell surfaces become spiked and ruffled. Cells fail to develop a furrow but, nevertheless, form zones of RhoA activation (arrowheads, 28:40). Bright, glowing objects in other blastomeres are nuclei that accumulate eGFP probes nonspecifically (see Fig. 2, C and D). (B) Projection of four sections through a purple urchin embryo to which 10 μM cytochalasin D was added shortly before furrowing. Furrows with associated RhoA activity develop (arrowheads) and regress, but equatorial RhoA activity remains high despite furrow regression. (C) Projection of 20 sections through the near surface of the embryo shown in B; although the cortex of cytochalasin D–treated cells is wildly deranged, RhoA remains elevated in the equator (arrowheads). (D) Projection of 15 sections through a green urchin embryo to which 10 μM cytochalasin D was added at metaphase. Zones of RhoA activity (arrowheads) appear on schedule despite the absence of ingression. (E) A more extreme case than D; projection of 10 1-μm sections through one blastomere of an eight-cell green urchin embryo attempting to cleave in 12-μM cytochalasin D. Active RhoA appears on tubular extensions projecting inward from the cortex, most prominently near the equator. (F) Projection of 38 sections through an eight-cell green urchin embryo treated identically to that in E. Tubular projections point toward the spindle poles (asterisks). See online supplemental material for videos corresponding to A (Video 6) and E (Video 7, available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1). Bars, 25 μm.
	Figure 5. Microtubule disruption disrupts RhoA zone. (A) Projection of seven sections through a purple urchin embryo, which was first treated with 12 μM cytochalasin D immediately before furrowing, and then treated with 25 μM nocodazole at the time the furrows began to regress (00:00). Nocodazole causes rapid abolition of RhoA zones (arrowheads). Bar, 25 μm. (B) Projection of 18 sections through purple urchin embryo cultured in 5 nM nocodazole for 30 min before filming. Furrows in top two cells are associated with uneven, poorly bounded zones of RhoA activity (arrowheads, 00:00). Zones subsequently fragment, and furrows regress. (C) Projection of 18 sections through a control purple urchin embryo showing normal width and brightness of the RhoA zone during telophase. (D) Projection of 18 sections through a four-cell purple urchin embryo cultured in 5 nM nocodazole. The RhoA zone is much wider than controls (compare brackets in C and D). (E) Scatter plot showing that RhoA zones are consistently wider in embryos treated with 5 nM nocodazole. Zones were measured at the point just after ingression begins (exemplified by the lower left furrows in C and D) in four untreated and six treated embryos of equivalent developmental stage (four and eight cell) from identical experiments on two successive days. Measurements from the same embryo are aligned vertically. Control and nocodazole-treated cells are significantly different (t test: P = 10−6).
	Figure 6. Physical displacement of the cortex relative to the spindle midplane modifies the RhoA zone. All times in this figure are in minutes:seconds relative to the physical perturbation indicated in the diagrams on the left. In each case, the dashed outlines in the first frame show the position of the spindle and the glass needle. Blastomeres from green urchin embryos at the 32–64-cell stage were used for these experiments. (A) Projection of four sections through a cell upon which the needle was pressed slightly to one side of the spindle, distending a portion of the cell into a pouch ∼10 μm thick. The side nearer the spindle initiates a tightly focused RhoA activity-rich furrow that ingresses (arrows); the far side accumulates active RhoA (brackets) but fails to focus it, and a series of shallow, unstable furrows develop and regress. (B) Projection of nine sections through a blastomere in which the spindle was displaced along the polar axis after furrow initiation. In the first frame, the blunt-ended needle is parked against one pole of the cell, slightly denting it. At 00:00, the needle was advanced by ∼15 μm (without penetrating the cell membrane), shoving the spindle so that the midplane shifts up relative to the furrow. Arrow and bracket in 00:20 mark the position immediately after the shove of the furrow and the RhoA zone, respectively, and remain the same for comparison in subsequent frames. The RhoA zone climbs higher on the cortex after the spindle midplane and is followed, in turn, by the furrow (second set of brackets and arrows in 03:00–06:40) such that the cell cleaves asymmetrically. (C) Projection of eight sections through a cell subjected to the converse of B: the cortex was displaced by a blunt needle toward the spindle midzone. In −00:20, the needle is parked slightly off center; at 00:00, the needle was advanced to bring a patch of nonequatorial cortex deep into the equatorial zone. At 06:20, needle tip shows accumulation of active RhoA (arrow). In addition, the original RhoA zone slides up the cortex (brackets in 04:00–08:40), apparently after half of the mitotic apparatus is broken by the needle. See online supplemental material for videos corresponding to A (Video 8) and B (Video 9, available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1). Bars, 25 μm.
	Figure 7. Each half of the mitotic apparatus can induce one zone of RhoA activation and a corresponding furrow. Times are in minutes:seconds after the onset of filming (which began within a minute after cutting); dashed outline indicates the position of the needle. (A) Projection of eight sections through a cell bisected during metaphase. Arrowheads in the first frame indicate the centrosomes, which soon disappear from view. At approximately the time this cell would have entered anaphase, one patch of cortex near the spindle half in each cell fragment accumulates eGFP-rGBD (arrows, 06:00). (B) Projection of 10 sections through a cell cut in anaphase. The left spindle half moved away from the cut, whereas the right spindle half remained closer to the cut edge. In the left half, active RhoA immediately accumulated on the cut face near the site where the cut passed through the spindle. Within minutes, however, the spindle half in the left fragment induced a new zone (arrows in second frame), which proceeded to ingress, forming a tripartite furrow. The right half underwent unequal cleavage in association with an asymmetric RhoA zone and furrow. See Video 10 (available at http://www.jcb.org/cgi/content/full/jcb.200501131/DC1). (C) Projection of 12 sections through a cell cut (as a control for A and B) such that the entire spindle (dashed outline, 00:00) remains in one of the two fragments. Only the spindle-containing fragment exhibited elevated RhoA activity (arrow) and a cleavage furrow. Bars, 25 μm.

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