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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.
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