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Figure 1. Cortical pRLC modulation during mitotic progression. Medial single-section confocal images of first cell cycle S. purpuratus zygotes. All cells were identically fixed and stained with Sytox green to reveal nuclei (green) and an antibody to serine19 pRLC (lavender). Identical microscope settings were used throughout for pRLC, but laser intensity was adjusted for Sytox green to make different nuclear condensation states discernable. Lavender arrowheads indicate the polar cortex of the nascent daughter cells, where pRLC reaccumulates before abscission. The white arrowhead indicates pRLC-depleted surface flanking the pRLC-enriched furrow. Bar, 25 μm.
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Figure 2. pRLC elevation at the equatorial cortex correlates with proximity to ncdz-resistant astral microtubule tips. Medial single-section confocal images of S. purpuratus zygotes, from one female, synchronously fertilized and allowed to develop to the stage indicated, then fixed and stained for pRLC (lavender) and tubulin (white). In each two-cell panel, the bottom cell was treated with 20 μM ncdz for 5 min before fixation (11°C), whereas the top cell was fixed without drug treatment. Arrows indicate the ncdz-resistant astral microtubules that appear during anaphase and elongate; pRLC accumulates to high levels near their tips (G). Cyan arrowheads mark bundled kinetochore microtubules (C–E), red arrowheads indicate interpolar microtubules (E–G and J), and green arrowheads indicate reforming telophase nuclei (H–K). Identical microscope settings were used throughout for pRLC. Laser intensity was adjusted to ensure visibility of microtubule tips, and higher laser intensity was used for the ncdz-treated cells (thus, e.g., stable kinetochore microtubules, represented by cyan arrowheads in C, appear brighter in ncdz-treated than in control cells). Bar, 20 μm.
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Figure 3. Prolonged ncdz treatment reveals complex gating of cortical pRLC levels. Medial single-section confocal images of S. purpuratus zygotes, from one female, fertilized synchronously, cultured to the stage shown, and immersed in 20 μM ncdz for the period indicated, then identically fixed and stained to reveal pRLC (lavender) and DNA (green). Identical microscope settings were used throughout for pRLC, but laser intensity was adjusted for different DNA condensation states. The number in each panel indicates chronological age (11.5°C) defined by the interval between sperm addition and the moment when that cell was transferred into fixative (e.g., 1:35 means fixed 1 h, 35 min after fertilization). The left column (0 min in 20 μM ncdz) shows the normal progression of control cells developing without ncdz. Each row displays cells that have spent 5, 10, and 20 min in ncdz, having been transferred into ncdz at the same time the control cells in the same row were transferred into fixation medium. Each panel shows one example of the most abundant phenotype for the specified time point, typically the only phenotype, determined by examining a slide containing >100 cells for the time point in question. Bar, 25 μm.
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Figure 4. The cortex reacts differently to the proximity of stable vs. dynamic microtubules. S. purpuratus zygotes: (A–C and G–I) Projections of 71 serial confocal sections (0.5 μm apart) revealing surface views of pRLC (white). Immediately below each surface projection are medial sectional views of the same cell (D–F and J–L) showing microtubules (white) and pRLC (lavender). (A–C) Telophase zygotes after 0, 4, and 8 min in ncdz (12°C) from the same preparations as Fig. S3, Q, S, and U, respectively (available at http://www.jcb.org/cgi/content/full/jcb.200807128/DC1). (G) An anaphase zygote, developing normally without ncdz, fixed just before elongating microtubules contacted the equator. (H and I) Late anaphase zygotes cultured in 1 mM colchicine. The same confocal microscope settings were used throughout for pRLC, but adjusted to optimize microtubule images. Bars, 25 μm; Surface projections are shown at 1.3× higher magnification than midsection micrographs.
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Figure 5. Equatorial recruitment of pRLC does not require F-actin. Surface views made from projecting 64 serial confocal sections (0.5 μm apart) of two sibling D. excentricus zygotes. Cells in B and D were cultured in 20 μM latrunculin (13°C); cells in A and C were cultured without latrunculin. Both cells were fixed during telophase. A and B show F-actin (stained with Bodipy FL phallacidin); C and D show pRLC. Identical preparation protocols and microscope settings were used for cells in A and B, and C and D, respectively. Bar, 35 μm.
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Figure 6. Recruitment of pRLC to the cell cortex requires Rho activity. Single medial confocal sections of fixed and stained D. excentricus embryos at first (A–F) and third (G) telophase. (A–C) Noninjected controls. (D–G) Zygotes injected with C3-transferase >15 min before NEB. (D–G) Coinjected fixable FITC-dextran labels the cytoplasm (to mark injected cells). A and D, B and E, and C and F are matched by mitotic phase. All cells were stained to show pRLC (lavender) and nuclei (green). Bar, 25 μm.
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Figure 7. Stable astral microtubules form without Rho activity. Medial single-section confocal images of D. excentricus zygotes. (A–D) Cells were treated with 20 μM ncdz for 5 min (12°C), then fixed and stained to reveal ncdz-resistant microtubules. (E–H) Cells were injected at least 15 min before NEB with C3, which induced an extended interphase. C3-injected cells were monitored under bright field until they resumed mitosis, transferred at the desired stage into 20 μM ncdz for 5 min (12°C), then fixed and stained identically to the noninjected cells. Shown are anaphase (A and B, and E and F), telophase (C and G), and early interphase (D and H). Arrowheads indicate condensed interpolar array, which is absent from same-stage C3-injected cells. Loss of dynamic astral microtubules frequently permits the interpolar array to slip sideways during ingression (as in D). Bar, 25 μm.
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