Field CM et al. (2019), Disassembly of Actin and Keratin Networks by Au...

XB-ART-56016

Curr Biol 2019 Jun 17;2912:1999-2008.e4. doi: 10.1016/j.cub.2019.05.016.

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Disassembly of Actin and Keratin Networks by Aurora B Kinase at the Midplane of Cleaving Xenopus laevis Eggs.

Field CM , Pelletier JF , Mitchison TJ .

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The large length scale of Xenopus laevis eggs facilitates observation of bulk cytoplasm dynamics far from the cortex during cytokinesis. The first furrow ingresses through the egg midplane, which is demarcated by chromosomal passenger complex (CPC) localized on microtubule bundles at the boundary between asters. Using an extract system, we found that local kinase activity of the Aurora B kinase (AURKB) subunit of the CPC caused disassembly of F-actin and keratin between asters and local softening of the cytoplasm as assayed by flow patterns. Beads coated with active CPC mimicked aster boundaries and caused AURKB-dependent disassembly of F-actin and keratin that propagated ∼40 μm without microtubules and much farther with microtubules present. Consistent with extract observations, we observed disassembly of the keratin network between asters in zygotes fixed before and during 1st cytokinesis. We propose that active CPC at aster boundaries locally reduces cytoplasmic stiffness by disassembling actin and keratin networks. Possible functions of this local disassembly include helping sister centrosomes move apart after mitosis, preparing a soft path for furrow ingression, and releasing G-actin from internal networks to build cortical networks that support furrow ingression.

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Species referenced: Xenopus laevis
Genes referenced: actb aurkb dnai1 incenp mapre1 nhs psmd6
GO keywords: spindle

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	Graphical Abstract
	Figure 1. AURKB-Dependent Disassembly of F-Actin at Boundaries between Asters in Egg Extract (A) Control reaction containing probes for F-actin (Lifeact-GFP), CPC (Alexa 647-anti-INCENP IgG), and microtubules (Tau peptide-mCherry) imaged by wide-field epifluorescence with a 20× objective. Note recruitment of CPC to anti-parallel microtubule bundles at boundaries between asters, and loss of F-actin bundles where CPC is recruited. Third row: higher-magnification views of the boxed region from the top left panel. Note the F-actin comet tails in the cleared region (chevrons, and 3× insets). See Videos S1 and S2. (B) Reaction containing 40 μM barasertib, a selective AURKB inhibitor. Asters grow and contact each other but CPC recruitment is blocked, and F-actin bundles are not disassembled at aster boundaries. Third row: higher-magnification views of the boxed region in the top left panel. See Video S3.
	Figure 2. Higher Spatiotemporal Resolution Imaging of F-Actin, CPC, and Microtubules during and after CPC-Positive Boundary Formation (A) Initiation of actin disassembly at a CPC-positive boundary in a reaction containing probes for F-actin (Lifeact-GFP), CPC (Alexa 647-anti-INCENP IgG), and microtubules (Tau peptide-mCherry) imaged by spinning-disk confocal microscopy with a 60× objective. Note alignment of the remaining F-actin bundles as the overall density decreases. Third row: higher-magnification views of actin in the boxed regions. (B) Later sequence in the same reaction at higher temporal resolution illustrating multiple F-actin comets growing in and around the boundary region. Third row: higher-magnification views in the boxed regions showing a typical F-actin comet growing in a boundary region where most F-actin bundles have disassembled. See also Videos S4 and S5.
	Figure 3. AURKB-Dependent Disassembly of Keratin at Boundaries between Asters in Egg Extract (A) Control reaction containing probes for keratin (Alexa 568-anti-keratin IgG), CPC (Alexa 647-anti-INCENP IgG), and growing microtubule plus ends (EB1-GFP). Note the partial disassembly of keratin at boundaries between asters where CPC is recruited. Keratin aggregates transiently accumulated at boundary lines, noticeable at 28 min in this example. See Video S6, which shows a different control experiment. (B) Images from an experiment similar to (A) run on a different day and chosen to highlight a boundary zone that formed later in the reaction, after the keratin network was established. Bulk flow caused the network to move leftward and upward in this time series. See Video S7. (C) Parallel reactions to (A) containing 40 μM barasertib CPC recruitment and keratin disassembly at aster boundaries are both blocked. Keratin clearing in a circular zone near MTOCs is similar to the control reaction in (A). Bottom row: higher-magnification views of the boxed region in the top left panel. Note the gradual evolution of the keratin signal from homogeneous aggregates into a network of connected bundles. Similar time-dependent assembly occurred in controls. (A) and (C) are from reactions run in parallel with Figure 1 using multi-position time lapse, so timing can be directly compared.
	Figure 4. Low Resistance to Pressure-Induced Flow between Asters (A) Flow in an aster assembly reaction similar to Figure 1A. Fluorescent images were collected immediately before initiating flow. Actin (Lifeact-GFP), tubulin (Tau peptide-mCherry), and CPC (anti-INCENP-Alexa 647). Right: a DIC still image taken from a 20-s movie collected immediately after inducing flow with a pressure transient. Cyan arrows indicate the velocity of particle movement, measured by particle image velocimetry (PIV) analysis of two sequential frames from the movie. Flow occurred preferentially through the F-actin-free channels between asters. Peak flow in this example was ∼7 μm/s. (B) Parallel reaction to (A) containing 40 μM barasertib to inhibit AURKB. AURKB does not localize between asters. Slower flow that was uniform across the whole field was observed following a pressure transient. Cyan arrows are at the same scale as in (A). See Video S8.
	Figure 5. Inhibition of Cytoskeleton Assembly around CPC Beads (A) F-actin clearing around 7-μm CPC beads in the absence of microtubules. t = 45 min. (B) F-actin was not cleared when AURKB activity was inhibited. t = 45 min. (C) Quantification of F-actin clearing from the experiment shown in (A) and (B). 15 beads were analyzed per condition. Dark lines show average radial intensity values; pale regions show the SD of intensity values. Linescans were truncated below 7 μm to avoid the strong bead edge signal. (D) Large zones of F-actin, microtubules, and keratin disassemble around 2.8-μm CPC beads when microtubules are present. t = 60 min. F-actin and microtubules disassembled in zones initially ∼50 μm in diameter, which grew progressively to 200 μm or more. Keratin disassembled over smaller diameters. See Video S9. (E) AURKB activity was required for disassembly. t = 60 min. See Table S1. (F) Microtubules were required for disassembly. t = 60 min. (G) EB1 comets were present throughout the extract at t = 60 min, and were lost around CPC beads. Conditions were similar to (D). (H) CPC-coated microtubule bundles around CPC beads. Conditions were similar to (D), except that Dasra-GFP was added to visualize CPC. t = 60 min. (I) Time-lapse sequence of formation of CPC-coated microtubule bundles and F-actin disassembly around CPC beads. Conditions were similar to (H). (A) and (B) are from parallel reactions. (D)–(F) are taken from parallel reactions in a different experiment. See Table S1. 7-μm (A and B) or 2.8-μm (C–I) protein A beads coated with anti-INCENP IgG were used to recruit and activate endogenous CPC in extract. 40 μM nocodazole and/or 40 μM barasertib were included, as indicated, to inhibit microtubules and/or AURKB. Yellow chevrons indicate CPC beads. Actin (Lifeact-GFP), keratin (Alexa 568-anti-keratin IgG), tubulin (directly labeled with Alexa 647 NHS ester), CPC (DasraA-GFP), and growing microtubule plus ends (EB1-GFP). See Videos S10 and S11.
	Figure 6. Keratin Disassembly ahead of Cleavage Furrow Ingression in Fixed Zygotes (A) 70 min post fertilization; the egg viewed from the animal pole. The asters have grown to ∼65% of the egg radius; cleavage has not yet initiated. Keratin bundles are not yet pronounced in the bulk cytoplasm. Keratin appears partly disassembled in an ∼30-μm zone at the aster boundary. Aster boundaries with a line of keratin fragments in the center are seen, resembling those seen in extract (Figure 2A). (B) 90 min post fertilization; the animal pole is at the top left. This zygote recently initiated cleavage. Keratin bundles are evident in the higher-magnification view (different focal plane). Note the partial clearing and accumulation of aggregates at the center of the aster boundary region, similar to (A). (C) 100 min post fertilization; the animal pole is at the top. The furrow has ingressed farther than (B), and keratin bundles are more pronounced away from the aster boundary. Large keratin bundles also accumulate at the cortex (not shown). Disassembly of the keratin network ahead of the ingressing furrow is evident, especially in the higher-magnification view. Xenopus zygotes were fixed 70–100 min post fertilization, bleached, stained, and imaged by confocal microscopy in a clearing solvent. The second image pair in each row is a 4× view of the boxed region.
	Figure 7. Model for CPC Recruitment and Functions at Aster Boundaries (A) Summary of molecular activities of the CPC. Red arrows indicate a positive feedback loop that promotes recruitment and spreading of the CPC on microtubule bundles at aster boundaries [2, 7]. Green and blue negative arrows indicate AURKB-dependent inhibition of all three cytoskeletal systems, presumably via reaction-diffusion mechanisms. (B–D) Illustration of possible functions of F-actin and keratin disassembly activities. (B) Disassembly of cytoskeleton networks between microtubule asters may help centrosomes and nuclei move apart following anaphase. (C) Disassembly of cytoskeleton networks may help steer the ingressing furrow. (D) Disassembly of bulk F-actin may supply the ingressing plasma membrane with subunits to build the new cortex, in a model where different F-actin assemblies compete for subunits.

References [+] :

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