Rho-dependent transfer of Citron-kinase to the cleavage furrow of dividingcells

Citron-kinase (Citron-K) is a Rho effector working in cytokinesis. It is en riched in cleavage furrow, but how Rho mobilizes Citron-K remains unknown. Using anti-Citron antibody and a Citron-K Green Fluorescence Protein (GFP)- fusion, we monitored its localization in cell cycle. We have found: (1) Cit ron-K is present as aggregates in interphase cells, disperses throughout th e cytoplasm in prometaphase, translocates to cell cortex in anaphase and ac cumulates in cleavage furrow in telophase; (2) Rho colocalizes with Citron- K in the cortex of ana- to telophase cells and the two proteins are concent rated in the cleavage furrow and to the midbody; (3) inactivation of Rho by C3 exoenzyme does not affect the dispersion of Citron-K in prometaphase, b ut prevented its transfer to the cell cortex, and Citron-K stays in associa tion with the midzone spindles of C3 exoenzyme-treated cells. To clarify fu rther the mechanism of the Rho-mediated transfer and concentration of Citro n-K in cleavage furrow, we expressed active Val14RhoA in interphase cells e xpressing GFP-Citron-K. Val14RhoA expression transferred Citron-K to the ve ntral cortex of interphase cells, where it formed bandlike structures in a complex with Rho. This structure was localized at the same plane as actin s tress fibers, and they exclude each other. Disruption of F-actin abolished the band and dispersed the Citron-K-Rho-containing patches throughout the c ell cortex. Similarly, in dividing cells, a structure composed of Rho and C itron-K in cleavage furrow excludes cortical actin cytoskeleton, and disrup tion of F-actin disperses Citron-K throughout the cell cortex. These result s suggest that Citron-K is a novel type of a passenger protein, which is di spersed to the cytoplasm in prometaphase and associated with midzone spindl es by a Rho-independent signal. Rho is then activated, binds to Citron-K an d translocates it to cell cortex, where the complex is then concentrated in the cleavage furrow by the action of actin cytoskeleton beneath the equato r of dividing cells.