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Fig. 3. Subcellular localisation of Citron-GFP to the contractile ring during cytokinesis depends on the normal activation of Rho signalling. (A-H) Citron-GFP was expressed in the 3-6 hour embryonic epidermis using paired-Gal4. DNA is stained with Hoechst 33258 (B,F), Citron-GFP is stained with anti-GFP (C,G), and microtubules are stained with anti--tubulin antibodies (D,H). (A,E) Merged images with Citron-GFP stained green, DNA stained blue and microtubules stained red. (A-D) A wild-type embryo showing Citron-GFP in the contractile ring (arrow) as it constricts around the central spindle microtubules. Citron-GFP is not localised in the adjacent anaphase cell that is yet to constrict (arrowhead). (E-G) An embryo mutant for the Rho activator pebble showing typical tetranucleate and binucleate cells and bipolar and tripolar spindles of cells failing cytokinesis. Citron-GFP shows no localisation to the contractile ring in pebble mutant telophase cells. It is found diffusely through the cytoplasm of telophase cells and not at the positions where contractile rings would normally form (arrowheads). (I-J) Drosophila Schneider line 2 cultured cells stained for DNA (green) and actin (red). Incubation with citron dsRNA (I) or dsRNA corresponding to the Rho activator pebble (J) causes the formation of multinucleate cells.

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