Field CM , Groen AC , Nguyen PA , Mitchison TJ .

	FIGURE 1: Cleavage pattern in a bispermic frog egg. Metaphase spindles in Xenopus are ∼50–60 μm long in a 1.2-mm egg. After anaphase onset, the small asters at the spindle poles grow steadily until they fill the cell. Asters from the two poles of the same spindle (sister asters) contact each other soon after anaphase onset at the plane previously occupied by the metaphase plate. Asters from the poles of two different spindles (nonsister asters) contact each other later. In both cases, aster growth halts where the asters contact each other, and a zone of lower microtubule density is observed at the aster–aster interaction zone (gray shading). When asters grow to touch the cortex, furrows initiate between sister aster pairs but not between nonsister pairs. Adapted from Brachet (1910), Wühr et al. (2009), Snook et al. (2011), and Mitchison et al. (2012).
	FIGURE 2: Localization of furrow-inducing protein complexes in polyspermic eggs. Eggs were fixed between first mitosis and first cleavage, stained for AurkB (a subunit of the CPC) and Kif23 (a subunit of Centralspindlin), and imaged by confocal microscopy. Cyan double-headed arrows illustrate sister aster pairs. (A) Normal, monospermic egg. Note plane of CPC- and Centralspindlin-positive microtubule bundles between sister asters. (B) Bispermic egg fixed a few minutes after anaphase onset. The asters have just started to grow. A CPC-positive disk is evident at the plane previously occupied by the metaphase plate, where sister asters recently met. Centralspindlin recruitment is less evident (cyan single arrow in Kif23 image). (C) Bispermic egg fixed midway between anaphase and cleavage. Interaction zones between sister and nonsister asters were evident in the tubulin image. Note a brighter tuft of microtubules that connects the sister pair to the upper left, in register with the centrosomes. This midzone-like morphological marker reveals the previous position of the spindle and was used to identify sister aster pairs in microtubule images. CPC and Centralspindlin have been recruited to the interaction zone between sister asters but not to the zone between nonsister asters. (D) Egg with four sperm fixed early in cleavage. Cleavage has initiated near the animal pole (not shown). This image shows a focal plane near the equator, where three aster pairs are visible. Furrowing had not yet occurred in this focal plane, but asters had grown all the way to the cortex. Note selective recruitment of CPC and Centralspindlin to zones between sister asters. (E) Highly polyspermic egg fixed early in cleavage. The region presented at higher magnification shows a single aster with CPC-positive zones at the junction with several neighboring asters (yellow arrowheads). None of these are zones with its sister aster, which is in a different z-plane.
	FIGURE 3: Forced activation of the CPC induces ectopic furrows. Monospermic eggs were injected after first mitosis (60–70 min postfertilization) and time-lapse imaged to score cleavage (A, C, E), or fixed around the time of first cleavage and stained for tubulin and AurkB for CPC (B, D, F). (A, B) Injection with an activating anti-INCENP IgG. (A) Note multiple ectopic furrows. (B) Note ectopic microtubule assemblies with CPC strongly localized to their peripheries. Furrows initiated where CPC planes touched the cortex (white arrows). (C, D) Injection with inhibitory IgG to MCAK (Kif2C), the predominant microtubule catastrophe factor in frog eggs. (C) Note multiple ectopic furrows. (D) Note ectopic, CPC-positive microtubule assemblies and associated ectopic furrows (white arrows) as in B. (E, F) Injection with buffer only. A single cleavage furrow forms (E and white arrow in F).
	FIGURE 4: Forced activation can recruit the CPC to interaction zones between nonsister asters. An activating anti-INCENP IgG was injected into polyspermic eggs 60–70 min postfertilization (after first mitosis). Sister asters in A–C are indicated by cyan double-headed arrows. (A) Bispermic egg fixed 90 min postfertilization. Note recruitment of CPC at similar levels to the interaction zones between sister and nonsister asters (CPC recruitment between nonsisters is highlighted by yellow arrowheads). Note that the aster morphology has been slightly distorted by the injection. (B) Bispermic egg fixed 98 min postfertilization. Same labeling convention as in A. CPC is recruited to interaction zones between nonsister asters. (C) Uninjected bispermic egg fixed at the same stage as A for comparison. CPC is not recruited between nonsister asters.
	FIGURE 5: Forced activation of the CPC promotes recruitment in the extract system. Interphase egg extracts containing fluorescence probes for tubulin and the CPC (directly labeled anti-AurkB) and beads coated with anti-AurkA as artificial centrosomes. (A) Representative images from time-lapse sequences collected in parallel from the same preparation of extract. Note that more CPC-positive zones were formed between nucleating sites in extracts treated with activating IgG to INCENP, Taxol (0.5 μM final), and inhibitory IgG to MCAK than in control. Once formed, CPC-positive zones were stable for the duration of experiments under all conditions except MCAK inhibition, where they formed early relative to control but then decayed. (B) Higher-magnification image sequences of the regions shown by red boxes in A. Regions were chosen to highlight the establishment and growth of CPC-positive zones between nucleating sites. Note that the zone from control and Taxol-treated extracts grows laterally after it initiates. CPC-positive zones formed earlier when the CPC was activated or microtubules were stabilized. (C) Quantification of CPC-positive zone formation from multiple image fields. At each time point, the length of CPC-positive zones in a field was measured and divided by the number of nucleating beads in the field. Note that CPC stimulation (anti-INCENP), Taxol, and MCAK inhibition all stimulated the rate and extent of CPC-positive zone formation. The last time point was omitted from the MCAK inhibition experiment because CPC-positive zones were unstable under this condition. With regard to A, see Supplemental Movies S5A Control and S5B Taxol.
	FIGURE 6: Initial distance between nucleating sites influences CPC recruitment. The same experiment as in Figure 5 was performed without molecular perturbations. The CPC probe is directly labeled anti-AurkB, and beads coated with anti-AurkA are the aster nucleators. Bead pairs were identified based on whether their asters interacted at the end of the movie. The distance between the bead pairs was measured at the start of the movie, and whether or not a CPC-positive zone formed between beads was scored upon initial aster–aster contact. (A) Example images from a time-lapse sequence. Arrowheads indicate typical bead pairs. Yellow and blue show examples where AurkB-positive zones did not form between beads; magenta shows examples where AurkB-positive zones did form. Note that the yellow arrowheads are close together (n = 2), blue arrowheads are further apart (n = 7), and magenta arrowheads are separated by an intermediate distance (n = 15). (B). Histogram of initial bead separations plotted separately for bead pairs where an AurkB-positive zone did (blue bars; n = 39) or did not (red bars; n = 30) form upon aster–aster contact. Initial distances were pooled for multiple fields imaged in parallel in two independent experiments (six fields total). Note that the initial separation distance was unimodal for bead pairs for which a CPC-positive zone did form. The distribution was much more spread out, and possibly bimodal, for bead pairs for which a CPC-positive zone did not form.
	FIGURE 7: Chromatin spatially biases CPC recruitment to monopolar sperm asters in egg extract. Permeabilized sperm was added to interphase egg extract supplemented with probes for DNA, tubulin, and the CPC (GFP-DasraA). (A–C) Image sequences chosen to emphasize different aspects of this reaction. The last in each series is a 3× magnification. Time points are as indicated. (A) Two isolated sperm asters. Note polarized microtubule distribution and formation of an arc of CPC-positive bundles on the periphery of each aster on the same side as the DNA. (A′) Only the DasraA channel for the same images as A. Note that the DasraA probe binds sperm chromatin. (A′′) Only the microtubule channel. (B) Field at higher sperm density. (B′) Same images in DasraA channel alone. Seven of eight of the asters polarize early (yellow p), and one aster never polarizes (cyan np). Later most asters contact a neighbor and form a CPC-positive interaction zone; yellow arrow at 11-min time point (B). (C) Field that includes a centrosome not attached to a sperm nucleus (white arrow in 12-min image). The centrosome nucleated a weaker aster than the sperm-attached centrosomes. The free centrosome aster did not recruit CPC to its periphery until it contacted a neighboring aster (30-min time point). The bright dot on the lower left side of the free centrosome aster is DNA without a centrosome. With regard to A–C, see Supplemental Movies 7A–7C, respectively.
	FIGURE 8: Model illustrating results and hypothetical underlying mechanisms. The image illustrates the situation in a bispermic egg between first anaphase and first cleavage, similar to the cartoon in Figure 1 and the experimental examples in Figures 2C and 4C. Color coding and orientation of sister and nonsister aster pairs are as in Figure 1; red boxes indicate CPC and Centralspindlin complexes. The text boxes linked by arrows are factors we identified that control CPC recruitment and activation. We hypothesize that locally acting signals from chromatin (Figure 7) and the starting distance between asters (Figure 6) together provide an initial bias that promotes CPC recruitment between sister asters and are lacking between nonsister asters. We further hypothesize that CPC activation/recruitment (Figures 3–5) and microtubule bundling/stabilization (Figures 3 and 5) together constitute a positive feedback loop that promotes CPC retention on microtubule bundles and lateral spreading of CPC-positive bundles. These positive feedbacks allow a CPC-positive interaction zone to grow radially from the site previously occupied by the metaphase plate all the way to the cortex while retaining its CPC-positive state.
	FIGURE 1:. Cleavage pattern in a bispermic frog egg. Metaphase spindles in Xenopus are ∼50–60 μm long in a 1.2-mm egg. After anaphase onset, the small asters at the spindle poles grow steadily until they fill the cell. Asters from the two poles of the same spindle (sister asters) contact each other soon after anaphase onset at the plane previously occupied by the metaphase plate. Asters from the poles of two different spindles (nonsister asters) contact each other later. In both cases, aster growth halts where the asters contact each other, and a zone of lower microtubule density is observed at the aster–aster interaction zone (gray shading). When asters grow to touch the cortex, furrows initiate between sister aster pairs but not between nonsister pairs. Adapted from Brachet (1910), Wühr et al. (2009), Snook et al. (2011), and Mitchison et al. (2012).
	FIGURE 2:. Localization of furrow-inducing protein complexes in polyspermic eggs. Eggs were fixed between first mitosis and first cleavage, stained for AurkB (a subunit of the CPC) and Kif23 (a subunit of Centralspindlin), and imaged by confocal microscopy. Cyan double-headed arrows illustrate sister aster pairs. (A) Normal, monospermic egg. Note plane of CPC- and Centralspindlin-positive microtubule bundles between sister asters. (B) Bispermic egg fixed a few minutes after anaphase onset. The asters have just started to grow. A CPC-positive disk is evident at the plane previously occupied by the metaphase plate, where sister asters recently met. Centralspindlin recruitment is less evident (cyan single arrow in Kif23 image). (C) Bispermic egg fixed midway between anaphase and cleavage. Interaction zones between sister and nonsister asters were evident in the tubulin image. Note a brighter tuft of microtubules that connects the sister pair to the upper left, in register with the centrosomes. This midzone-like morphological marker reveals the previous position of the spindle and was used to identify sister aster pairs in microtubule images. CPC and Centralspindlin have been recruited to the interaction zone between sister asters but not to the zone between nonsister asters. (D) Egg with four sperm fixed early in cleavage. Cleavage has initiated near the animal pole (not shown). This image shows a focal plane near the equator, where three aster pairs are visible. Furrowing had not yet occurred in this focal plane, but asters had grown all the way to the cortex. Note selective recruitment of CPC and Centralspindlin to zones between sister asters. (E) Highly polyspermic egg fixed early in cleavage. The region presented at higher magnification shows a single aster with CPC-positive zones at the junction with several neighboring asters (yellow arrowheads). None of these are zones with its sister aster, which is in a different z-plane.
	FIGURE 3:. Forced activation of the CPC induces ectopic furrows. Monospermic eggs were injected after first mitosis (60–70 min postfertilization) and time-lapse imaged to score cleavage (A, C, E), or fixed around the time of first cleavage and stained for tubulin and AurkB for CPC (B, D, F). (A, B) Injection with an activating anti-INCENP IgG. (A) Note multiple ectopic furrows. (B) Note ectopic microtubule assemblies with CPC strongly localized to their peripheries. Furrows initiated where CPC planes touched the cortex (white arrows). (C, D) Injection with inhibitory IgG to MCAK (Kif2C), the predominant microtubule catastrophe factor in frog eggs. (C) Note multiple ectopic furrows. (D) Note ectopic, CPC-positive microtubule assemblies and associated ectopic furrows (white arrows) as in B. (E, F) Injection with buffer only. A single cleavage furrow forms (E and white arrow in F).
	FIGURE 4:. Forced activation can recruit the CPC to interaction zones between nonsister asters. An activating anti-INCENP IgG was injected into polyspermic eggs 60–70 min postfertilization (after first mitosis). Sister asters in A–C are indicated by cyan double-headed arrows. (A) Bispermic egg fixed 90 min postfertilization. Note recruitment of CPC at similar levels to the interaction zones between sister and nonsister asters (CPC recruitment between nonsisters is highlighted by yellow arrowheads). Note that the aster morphology has been slightly distorted by the injection. (B) Bispermic egg fixed 98 min postfertilization. Same labeling convention as in A. CPC is recruited to interaction zones between nonsister asters. (C) Uninjected bispermic egg fixed at the same stage as A for comparison. CPC is not recruited between nonsister asters.
	FIGURE 5:. Forced activation of the CPC promotes recruitment in the extract system. Interphase egg extracts containing fluorescence probes for tubulin and the CPC (directly labeled anti-AurkB) and beads coated with anti-AurkA as artificial centrosomes. (A) Representative images from time-lapse sequences collected in parallel from the same preparation of extract. Note that more CPC-positive zones were formed between nucleating sites in extracts treated with activating IgG to INCENP, Taxol (0.5 μM final), and inhibitory IgG to MCAK than in control. Once formed, CPC-positive zones were stable for the duration of experiments under all conditions except MCAK inhibition, where they formed early relative to control but then decayed. (B) Higher-magnification image sequences of the regions shown by red boxes in A. Regions were chosen to highlight the establishment and growth of CPC-positive zones between nucleating sites. Note that the zone from control and Taxol-treated extracts grows laterally after it initiates. CPC-positive zones formed earlier when the CPC was activated or microtubules were stabilized. (C) Quantification of CPC-positive zone formation from multiple image fields. At each time point, the length of CPC-positive zones in a field was measured and divided by the number of nucleating beads in the field. Note that CPC stimulation (anti-INCENP), Taxol, and MCAK inhibition all stimulated the rate and extent of CPC-positive zone formation. The last time point was omitted from the MCAK inhibition experiment because CPC-positive zones were unstable under this condition. With regard to A, see Supplemental Movies S5A Control and S5B Taxol.
	FIGURE 6:. Initial distance between nucleating sites influences CPC recruitment. The same experiment as in Figure 5 was performed without molecular perturbations. The CPC probe is directly labeled anti-AurkB, and beads coated with anti-AurkA are the aster nucleators. Bead pairs were identified based on whether their asters interacted at the end of the movie. The distance between the bead pairs was measured at the start of the movie, and whether or not a CPC-positive zone formed between beads was scored upon initial aster–aster contact. (A) Example images from a time-lapse sequence. Arrowheads indicate typical bead pairs. Yellow and blue show examples where AurkB-positive zones did not form between beads; magenta shows examples where AurkB-positive zones did form. Note that the yellow arrowheads are close together (n = 2), blue arrowheads are further apart (n = 7), and magenta arrowheads are separated by an intermediate distance (n = 15). (B). Histogram of initial bead separations plotted separately for bead pairs where an AurkB-positive zone did (blue bars; n = 39) or did not (red bars; n = 30) form upon aster–aster contact. Initial distances were pooled for multiple fields imaged in parallel in two independent experiments (six fields total). Note that the initial separation distance was unimodal for bead pairs for which a CPC-positive zone did form. The distribution was much more spread out, and possibly bimodal, for bead pairs for which a CPC-positive zone did not form.
	FIGURE 7:. Chromatin spatially biases CPC recruitment to monopolar sperm asters in egg extract. Permeabilized sperm was added to interphase egg extract supplemented with probes for DNA, tubulin, and the CPC (GFP-DasraA). (A–C) Image sequences chosen to emphasize different aspects of this reaction. The last in each series is a 3× magnification. Time points are as indicated. (A) Two isolated sperm asters. Note polarized microtubule distribution and formation of an arc of CPC-positive bundles on the periphery of each aster on the same side as the DNA. (A′) Only the DasraA channel for the same images as A. Note that the DasraA probe binds sperm chromatin. (A′′) Only the microtubule channel. (B) Field at higher sperm density. (B′) Same images in DasraA channel alone. Seven of eight of the asters polarize early (yellow p), and one aster never polarizes (cyan np). Later most asters contact a neighbor and form a CPC-positive interaction zone; yellow arrow at 11-min time point (B). (C) Field that includes a centrosome not attached to a sperm nucleus (white arrow in 12-min image). The centrosome nucleated a weaker aster than the sperm-attached centrosomes. The free centrosome aster did not recruit CPC to its periphery until it contacted a neighboring aster (30-min time point). The bright dot on the lower left side of the free centrosome aster is DNA without a centrosome. With regard to A–C, see Supplemental Movies 7A–7C, respectively.
	FIGURE 8:. Model illustrating results and hypothetical underlying mechanisms. The image illustrates the situation in a bispermic egg between first anaphase and first cleavage, similar to the cartoon in Figure 1 and the experimental examples in Figures 2C and 4C. Color coding and orientation of sister and nonsister aster pairs are as in Figure 1; red boxes indicate CPC and Centralspindlin complexes. The text boxes linked by arrows are factors we identified that control CPC recruitment and activation. We hypothesize that locally acting signals from chromatin (Figure 7) and the starting distance between asters (Figure 6) together provide an initial bias that promotes CPC recruitment between sister asters and are lacking between nonsister asters. We further hypothesize that CPC activation/recruitment (Figures 3–5) and microtubule bundling/stabilization (Figures 3 and 5) together constitute a positive feedback loop that promotes CPC retention on microtubule bundles and lateral spreading of CPC-positive bundles. These positive feedbacks allow a CPC-positive interaction zone to grow radially from the site previously occupied by the metaphase plate all the way to the cortex while retaining its CPC-positive state.

Argiros, Centralspindlin and chromosomal passenger complex behavior during normal and Rappaport furrow specification in echinoderm embryos. 2012, Pubmed

Argiros, Centralspindlin and chromosomal passenger complex behavior during normal and Rappaport furrow specification in echinoderm embryos. 2012, Pubmed
Baruni, Cytokinetic furrowing in toroidal, binucleate and anucleate cells in C. elegans embryos. 2008, Pubmed
Basant, Aurora B kinase promotes cytokinesis by inducing centralspindlin oligomers that associate with the plasma membrane. 2015, Pubmed
Bieling, A minimal midzone protein module controls formation and length of antiparallel microtubule overlaps. 2010, Pubmed , Xenbase
Canman, Determining the position of the cell division plane. 2003, Pubmed
Carmena, The chromosomal passenger complex (CPC): from easy rider to the godfather of mitosis. 2012, Pubmed
Cooke, The inner centromere protein (INCENP) antigens: movement from inner centromere to midbody during mitosis. 1987, Pubmed
Delacour-Larose, Distinct dynamics of Aurora B and Survivin during mitosis. 2004, Pubmed
Desai, The use of Xenopus egg extracts to study mitotic spindle assembly and function in vitro. 1999, Pubmed , Xenbase
Eggert, Animal cytokinesis: from parts list to mechanisms. 2006, Pubmed
Ferreira, Aurora B spatially regulates EB3 phosphorylation to coordinate daughter cell adhesion with cytokinesis. 2013, Pubmed
Field, Xenopus egg cytoplasm with intact actin. 2014, Pubmed , Xenbase
Foe, Stable and dynamic microtubules coordinately shape the myosin activation zone during cytokinetic furrow formation. 2008, Pubmed
Fuller, Midzone activation of aurora B in anaphase produces an intracellular phosphorylation gradient. 2008, Pubmed , Xenbase
Glotzer, The molecular requirements for cytokinesis. 2005, Pubmed
Gruneberg, Relocation of Aurora B from centromeres to the central spindle at the metaphase to anaphase transition requires MKlp2. 2004, Pubmed
Hu, Cell polarization during monopolar cytokinesis. 2008, Pubmed
Hu, KIF4 regulates midzone length during cytokinesis. 2011, Pubmed
Ishihara, Microtubule nucleation remote from centrosomes may explain how asters span large cells. 2014, Pubmed , Xenbase
Kelly, Chromosomal enrichment and activation of the aurora B pathway are coupled to spatially regulate spindle assembly. 2007, Pubmed , Xenbase
Kitagawa, Cdk1 coordinates timely activation of MKlp2 kinesin with relocation of the chromosome passenger complex for cytokinesis. 2014, Pubmed
Kitagawa, The chromosomal passenger complex (CPC) as a key orchestrator of orderly mitotic exit and cytokinesis. 2015, Pubmed
Lewellyn, The chromosomal passenger complex and centralspindlin independently contribute to contractile ring assembly. 2011, Pubmed
Maresca, Methods for studying spindle assembly and chromosome condensation in Xenopus egg extracts. 2006, Pubmed , Xenbase
Mitchison, Self-organization of stabilized microtubules by both spindle and midzone mechanisms in Xenopus egg cytosol. 2013, Pubmed , Xenbase
Mitchison, Growth, interaction, and positioning of microtubule asters in extremely large vertebrate embryo cells. 2012, Pubmed , Xenbase
Murata-Hori, Both midzone and astral microtubules are involved in the delivery of cytokinesis signals: insights from the mobility of aurora B. 2002, Pubmed
Murray, Cell cycle extracts. 1991, Pubmed
Nguyen, Spatial organization of cytokinesis signaling reconstituted in a cell-free system. 2014, Pubmed , Xenbase
Nunes Bastos, Aurora B suppresses microtubule dynamics and limits central spindle size by locally activating KIF4A. 2013, Pubmed
Ohi, Differentiation of cytoplasmic and meiotic spindle assembly MCAK functions by Aurora B-dependent phosphorylation. 2004, Pubmed , Xenbase
Rappaport, Aster-equatorial surface relations and furrow establishment. 1969, Pubmed
RAPPAPORT, Experiments concerning the cleavage stimulus in sand dollar eggs. 1961, Pubmed
Render, Axis determination in polyspermic Xenopus laevis eggs. 1986, Pubmed , Xenbase
Rieder, Mitosis in vertebrate somatic cells with two spindles: implications for the metaphase/anaphase transition checkpoint and cleavage. 1997, Pubmed
Savoian, Cleavage furrows formed between centrosomes lacking an intervening spindle and chromosomes contain microtubule bundles, INCENP, and CHO1 but not CENP-E. 1999, Pubmed
Snook, The biology and evolution of polyspermy: insights from cellular and functional studies of sperm and centrosomal behavior in the fertilized egg. 2011, Pubmed
Tsai, Aurora A kinase-coated beads function as microtubule-organizing centers and enhance RanGTP-induced spindle assembly. 2005, Pubmed , Xenbase
Tseng, Dual detection of chromosomes and microtubules by the chromosomal passenger complex drives spindle assembly. 2010, Pubmed , Xenbase
Tsuchiya, Apoptosis-inhibiting activities of BIR family proteins in Xenopus egg extracts. 2005, Pubmed , Xenbase
Walczak, XKCM1: a Xenopus kinesin-related protein that regulates microtubule dynamics during mitotic spindle assembly. 1996, Pubmed , Xenbase
White, Centralspindlin: at the heart of cytokinesis. 2012, Pubmed
Wühr, Deep proteomics of the Xenopus laevis egg using an mRNA-derived reference database. 2014, Pubmed , Xenbase
Wühr, How does a millimeter-sized cell find its center? 2009, Pubmed , Xenbase
Zimniak, Phosphoregulation of the budding yeast EB1 homologue Bim1p by Aurora/Ipl1p. 2009, Pubmed