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First published online 13 December 2005
doi: 10.1242/jcs.02737

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Centralspindlin regulates ECT2 and RhoA accumulation at the equatorial cortex during cytokinesis

Laboratory for Cellular Morphogenesis, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan

View larger version (38K): [in a new window]   Fig. 1. Localization of RhoA and DNA during cytokinesis in HeLa cells. In interphase, RhoA (upper panels) distributes diffusely in the cytoplasm. After onset of mitosis, RhoA begins to accumulate at the cell cortex (metaphase). Accumulation of RhoA at the equatorial cortex is detected before furrowing (arrowhead) and after which it forms a ring structure at the cleavage furrow (anaphase to telophase). Finally, RhoA localizes to the midbody (late telo.). Lower panels show DNA staining. Bar, 10 µm.

View larger version (31K): [in a new window]   Fig. 2. RhoA activity and microtubule organization are required for RhoA localization at the equatorial cortex. (A-C) Quantification of RhoA density at the equatorial cortex during metaphase or anaphase in HeLa cells. (A) Typical RhoA immunofluorescence images at metaphase (meta.) and anaphase (ana.). (B) Schematic drawing of cells used to measure RhoA fluorescence intensity. Stars represent points for measuring RhoA density at the cell pole and the equator. Dark ovals represent nuclei. (C) Fluorescence intensities of RhoA at the pole and the equator. RhoA density at the equator significantly increases after metaphase (n=10). (D) RhoA activity is required for localization at the equatorial cortex. HeLa cells were injected with Botulinum C3 exoenzyme (C3), RhoGDI or fluorescein dextran (control) before metaphase and incubated until control cells entered anaphase. No RhoA accumulation is evident in C3- or RhoGDI-injected cells. (E) Microtubule organization is required for RhoA localization at the equatorial cortex during cytokinesis. HeLa cells were treated with 200 µM latrunculin A or with 100 µM blebbistatin before metaphase onset and incubated until control cells entered anaphase. RhoA normally accumulated at the equatorial cortex (+LAT-A, + blebb., arrowheads). A6 cells expressing ß-tubulin-GFP were incubated with 16 µM nocodazole just after anaphase onset for 30 minutes. RhoA did not localize at the equatorial cortex but scattered on the cortex (+ Noc., arrowheads). Bars, 10 µm.

View larger version (52K): [in a new window]   Fig. 3. Microtubule organization is required for stable furrow progression and maintenance of RhoA localization. (A) Selected frames of Movie 1 in supplementary material showing phase-contrast images (upper panel) and ß-tubulin images (lower panel) of A6 cells expressing ß-tubulin-GFP. At initiation of furrowing when RhoA accumulated at the equatorial cortex, 16 µM nocodazole was added to the medium. Chromosomes are indicated by white arrows. A furrow (black arrows) formed and ingressed (3:20). But it quickly regressed (3:40-4:00). Then another furrow (white arrowheads) formed at a different position and ingressed (4:20-4:50). The furrow also regressed. This cell did not complete cytokinesis within 30 minutes. The elapsed time (minutes and seconds) from nocodazole treatment is indicated at the lower left corner of each panel. (B) The same cell as in A was fixed at 30 minutes of nocodazole application and stained for RhoA and DNA. RhoA localization was not restricted to the equatorial cortex but was found at several sites on the cortex (upper panel, arrowheads). Failure in cytokinesis is revealed by the formation of two nuclei (see DNA in lower panel). Bar, 10 µm.

View larger version (87K): [in a new window]   Fig. 4. Astral and central spindle microtubules can localize RhoA at the cell cortex. (A) Control and 150 nM nocodazole-treated NRK-52E cells at anaphase stained for RhoA (upper panels and magenta in lower panels and images 1-4) and -tubulin (green in lower panels and images 1-4). Upper and lower panels, X-Y (horizontal) images of cells projected to one plane from serial optical sections. Images 1-4 represent X-Z (vertical) images projected to one plane from serial reconstructed sections from the squares highlighted in the lower images, showing cortical localization of RhoA. Red arrowheads indicate cell equators. In cells treated with this dose of nocodazole, astral microtubules were selectively disrupted. RhoA accumulation is not evident at the peripheral equatorial cortex without astral microtubules (image 3) but is clear around the cell center near central spindle microtubules (image 4). (B) Quantification of RhoA accumulation in control and low-dose nocodazole-treated cells. RhoA accumulation is expressed as ratio of fluorescence intensity at the equatorial cortex in the cell center (white and dark gray bars) or in the cell periphery (light gray and black bars) over the fluorescence intensity at the polar cortex. In nocodazole-treated cells, RhoA accumulation is significantly reduced at the equatorial cortex near the cell periphery (black bar). Values are means ± s.e.m. of three images. (C) Central spindle microtubules induce partial contractile ring formation in nocodazole-treated NRK-52E cells. Phosphorylated myosin light chain (p-MLC) and F-actin are accumulated (lower panels, yellow arrows) along the equatorial cortex at the cell center close to central spindle microtubules (arrowheads in tubulin panels) in nocodazole-treated cells. DNA was also stained. Dotted lines in lower images indicate cell peripheries. (D) RhoA localization is normal in cells with disrupted central spindle microtubules. Left panels, PRC1 staining in HeLa cells. Middle panels, merged images with PRC1 (magenta) and -tubulin (green). Right panels, merged images of RhoA (magenta) and -tubulin (green). PRC1 is localized at central spindle microtubules in control HeLa cells. PRC1 depletion (PRC1 RNAi) resulted in disruption of central spindle microtubules. RhoA accumulation (yellow arrowheads) was normal in PRC1-depleted cells. All images in C and D are X-Y images projected to one plane from serial optical sections. Bars, 15 µm.

View larger version (29K): [in a new window]   Fig. 5. ECT2, HsCYK4 and MKLP1 are required for RhoA localization during cytokinesis. (A) Depletion of ECT2, HsCYK4 and MKLP1 was confirmed by immunoblotting with each antibody. Control HeLa cells treated with a scrambled siRNA (C) and cells depleted of each protein by RNAi (RNAi). GAPDH was detected as a loading control. (B) RhoA disappears from the equatorial cortex during cytokinesis in cells depleted of ECT2 (ECT2 RNAi), HsCYK4 (HsCYK4 RNAi) or MKLP1 (MKLP1 RNAi). Cells were stained with antibodies to RhoA and -tubulin and DAPI. All images are X-Y images of cells projected to one plane from serial optical sections. (C) Quantification of RhoA accumulation in control and ECT2-, HsCYK4-, and MKLP1-depleted HeLa cells. RhoA accumulation is expressed as ratio of fluorescence intensity at the equatorial cortex over the fluorescence intensity at the polar cortex. Stars on the schematic drawing of cells (right) represent points for measuring RhoA intensity at the cell pole and the equator. In ECT2 (light grey bar; n=35), HsCYK4 (dark grey bar; n=36) and MKLP1 (black bar; n=39) siRNA-treated cells, RhoA accumulation is significantly reduced at the equatorial cortex compared with that in cells treated with scrambled siRNA (control; white bar; n=20). Bar, 15 µm.

View larger version (99K): [in a new window]   Fig. 6. Localization of HsCYK4 and MKLP1 in HeLa cells. (A) HsCYK4 and MKLP1 localize to central spindle microtubules and the tips of astral microtubules in control cells (control) but not in siRNA-treated cells (RNAi). RhoA (red, in middle panels) accumulated at the equatorial cortex (arrows) in control cells. (B) Magnified images of the boxed areas of A. Arrows indicate tips of the astral microtubule (green) with accumulated HsCYK4 or MKLP1 (magenta). All images in A and B are X-Y images projected to one plane from serial optical sections. Bars, 15 µm (A); 5 µm (B).

View larger version (40K): [in a new window]   Fig. 7. Relationship between centralspindlin (HsCYK4/MKLP1) and ECT2. (A) HsCYK4 and MKLP1 regulate localization of ECT2 during cytokinesis. Localization of HsCYK4 and MKLP1 was not affected by ECT2 depletion (ECT2 RNAi, arrows). Compared with control cells (ECT2 control), ECT2 localization was lost in HsCYK4- or MKLP1-depleted cells (HsCYK4 RNAi, MKLP1 RNAi, arrows). All images are X-Y images projected to one plane from serial optical sections. (B) ECT2 forms a complex with MKLP1 and HsCYK4 in HeLa cells. As indicated above each lane, cell lysates were immunoprecipitated with anti-MKLP1, anti-HsCYK4 or anti-ECT2 antibodies. As a control, rabbit (R) and goat (G) IgG was used. Immunoprecipitates were then analyzed by western blotting with antibodies represented at the left side of each panel. MKLP1, HsCYK4 and ECT2 were detected in the immunoprecipitates of anti-MKLP1, anti-HsCYK4 and anti-ECT2 (black arrowheads), indicating that these proteins form a complex in vivo. Bar, 15 µm.

View larger version (40K): [in a new window]   Fig. 8. A model of RhoA localization at the equatorial cortex. HsCYK4 and MKLP1 (centralspindlin) localize both at the tips of astral microtubules towards the equator and at central spindle microtubules and recruit ECT2 at the equatorial cortex and central spindle microtubules. ECT2 then induces RhoA accumulation by elevating RhoA activity.