Mitchison TJ and Field CM (2017), Spindle-to-Cortex Communication in Cleaving Fro...

XB-ART-54328

Cold Spring Harb Symp Quant Biol 2017 Jan 01;82:165-171. doi: 10.1101/sqb.2017.82.033654.

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Spindle-to-Cortex Communication in Cleaving Frog Eggs.

Mitchison TJ , Field CM .

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During cytokinesis, the mitotic spindle communicates with the cell cortex to position a cleavage furrow that will cut through the cell in the plane defined by the metaphase plate. We investigated the molecular basis of this communication in Xenopus laevis eggs, where the signal has to travel ∼400 µm in ∼30 min to reach the cortex from the first anaphase spindle. At anaphase onset, huge microtubule asters grow out from the poles of the spindle and meet at the plane previously defined by the metaphase plate. This disc-shaped boundary plane recruits the chromosome passenger complex (CPC) and centralspindlin to antiparallel microtubule bundles. It grows out to the cell cortex as the asters expand, where it induces the furrow. CPC and centralspindlin were not recruited to boundaries between asters from different spindles, suggesting a role of chromatin in triggering the CPC-positive state. Recruitment of CPC to aster boundaries was reconstituted in an extract system, and we observed that recruitment was stimulated by proximity to chromatin. Finally, we discuss models for molecular processes involved in initiation and growth of the CPC-positive disc that communicates the position of the metaphase plate to the cortex over hundreds of micrometers in frog eggs.

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Species referenced: Xenopus laevis
Genes referenced: aurka aurkb frzb2 kif23 kif4a
GO keywords: spindle [+]

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	Figure 1. Spindle-to-cortex communication in different systems. (A) In most animal cells the spindle sends a signal to localize the cleavage furrow, which then cuts through the cell in the plane defined by the earlier metaphase plate. (B) In mechanically deformed echinoderm eggs, aster pairs trigger furrows whether they grow from the same or different spindles. This shows that chromatin is not essential to general the furrow signal. (C) In polyspermic frog eggs, only aster pairs from the same spindle trigger furrows. These images are drawn from sections of fixed frog eggs after polyspermic fertilization. The left egg was fixed before first mitosis illustrating five sperm asters. Black trails mark the path of the sperm as it moves into the cytoplasm after entering the egg. The right egg was fixed during first cleavage. It illustrates cleavage with five spindles, where each furrow cuts through the sites previously occupied by a metaphase plate. Note the lack of furrows between spindles, unlike the Echinoderm system. (B, Redrawn from Rappaport and Conrad 1963, with estimated chromatin localization added in blue; C, modified from Brachet 1910.)
	Figure 2. Aster growth and recruitment of furrow-stimulating complexes in Xenopus laevis eggs. Images are confocal sections viewed with the animal–vegetal axis in the microscope z-axis. The first cleavage furrow cuts through the animal pole, parallel to the z-axis in these images. (A,A′) Egg fixed ∼60 min after fertilization, at metaphase of first mitosis. Note the spindle is small compared to the egg. (B, C) Eggs fixed ∼70 and ∼80 min after fertilization, between first anaphase and first cleavage. Note aster pairs grow out from the poles of the spindle and meet at the midplane. Each aster is dome-shaped, with a sharp plane of lower microtubule density where they meet. In cross section this geometry appears as two D-shaped asters arranged back-to-back with a line between them. (D) Egg fixed at cleavage initiation, ∼90 min after fertilization. The asters are just touching the cortex at the animal pole. AurkB is a subunit of the CPC; Kif23 is a subunit of centralspindlin. Both furrow-stimulating complexes are enriched at a disc between the asters that marks the future path of the cleavage furrow. Note the centrosomes are already positioned for the second round of mitosis and cleavage that will occur orthogonal to the first (3× inset in tubulin panel). (E) Higher-magnification view of the boundary region between asters. Note the CPC localizes to the center of presumably antiparallel microtubule bundles. (A–C, Modified, with permission, from Mitchison et al. 2012; D, modified, with permission, from Field et al. 2015; E, TJ Mitchison and CM Field, unpubl. data.)
	Figure 3. Furrow-stimulating complexes in polyspermic Xenopus laevis eggs. Eggs fixed at successive times between first anaphase and first cleavage, with confocal sections oriented as in Figure 2. Cleavage has just initiated at the animal pole in C. The eggs in A and B were fertilized with two sperm and in C with four, but asters are only visible for three. Note recruitment of CPC and centralspindlin to a subset of boundaries between asters in B and C. In B, the aster morphology shows that the CPC-positive boundaries are between asters from the same spindle. In C, we infer the same is true given classic evidence that furrows cut through spindles in frog eggs and not between them (Fig. 1C). (Modified, with permission, from Field et al. 2015.
	Figure 4. Recruitment of CPC to aster boundaries in egg extract. Actin-intact extract from unfertilized eggs was supplemented with imaging probes, converted to interphase with a calcium transient, and squashed between passivated coverslips. (A) Widefield imaging of asters nucleated by anti-AurkA coated beads. CPC was visualized with anti-AurkB. Note recruitment of CPC to antiparallel bundles at the boundary between asters. (B) Total internal reflection fluorescence (TIRF) imaging of CPC movement in reactions similar to A. Movies were recorded at CPC-positive boundaries between asters, and kymograph analysis was used to visualize movement of CPC aggregates. The pink line denotes the center of the boundary region. Diagonal streaks represent movement of CPC aggregates toward plus ends at ∼15 µm/min. CPC recruitment to microtubules depended on an egg ortholog of Kif20Awe called Kif20AE, and movement depended on Kif4A as shown by this depletion-add back experiment. (C) Widefield imaging of asters nucleated by sperm centrosomes. CPC was imaged with GFP-DasraB. Note formation of polarized monopolar asters with CPC recruited to a crescent at the periphery. The CPC-positive crescent always formed on the chromatin-proximal side of asters and usually expanded radially as the asters grew. (A,B, Modified, with permission, from Nguyen et al. 2014; C, modified, with permission, from Field et al. 2015.)
	Figure 5. Working model for spindle-to-cortex communication in frog eggs. (A) Late-anaphase spindle morphology, shortly after initiating spindle-to-cortex communication. The spindle image is taken from the earliest time point in Figure 3. Arrows indicate directions of movement/growth. Note that the furrow-stimulating CPC-positive disc (red arrows) grows outward on a plane orthogonal to the axis of chromosome separation (blue arrows). (B) Models for the molecular events involved in triggering formation of the CPCpositive disc near chromatin (left), growing it outward (middle), and signaling to the cortex (right).

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