Fig. 4. Colocalization of RhoA and Citron-K in cleavage furrow of HeLa cells. Colocalization of Rho and Citron-K was examined by staining endogenous Rho with anti-Rho antibody (red, right-hand panels) in HeLa cells expressing GFP-Citron-K (green, middle panels) in various stages of cytokinesis. Left-hand panels represent merged images. In interphase, Rho shows homogenous staining in the cytoplasm, whereas Citron-K shows particulate signals (A). Punctate signals for Rho in the nucleus appear nonspecific, because such signals are only found by this polyclonal antibody 119 used in this experiment, and not by the other monoclonal antibody 26C4. GFP-Citron-K and endogenous RhoA colocalize around the cell equator of cells in the early stage (B), in the ingressing cleavage furrow in the middle stage (C), and are concentrated together at the cleavage site in the end stage (D) of cytokinesis. In addition, a portion of GFP-Citron-K remains as aggregates in the cytoplasm, where no colocalization with Rho was observed (for example, see arrowheads in B and C). Confocal sections of each cell are shown. Note that Citron-K and a part of Rho also colocalize in the midbody of post mitotic cells (E). Blue in E is ß-tubulin. Bars, 10 µm.