Rho-dependent transfer of Citron-kinase to the cleavage furrow of dividing cells.
Citron-kinase (Citron-K) is a Rho effector working in cytokinesis. It is enriched 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) Citron-K is present as aggregates in interphase cells, disperses throughout the cytoplasm in prometaphase, translocates to cell cortex in anaphase and accumulates in cleavage furrow in telophase; (2) Rho colocalizes with Citron-K in the cortex of ana- to telophase cells and the two proteins are concentrated 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, but prevented its transfer to the cell cortex, and Citron-K stays in association with the midzone spindles of C3 exoenzyme-treated cells. To clarify further the mechanism of the Rho-mediated transfer and concentration of Citron-K in cleavage furrow, we expressed active Val14RhoA in interphase cells expressing GFP-Citron-K. Val14RhoA expression transferred Citron-K to the ventral cortex of interphase cells, where it formed band-like structures in a complex with Rho. This structure was localized at the same plane as actin stress fibers, and they exclude each other. Disruption of F-actin abolished the band and dispersed the Citron-K-Rho-containing patches throughout the cell cortex. Similarly, in dividing cells, a structure composed of Rho and Citron-K in cleavage furrow excludes cortical actin cytoskeleton, and disruption of F-actin disperses Citron-K throughout the cell cortex. These results suggest that Citron-K is a novel type of a passenger protein, which is dispersed to the cytoplasm in prometaphase and associated with midzone spindles by a Rho-independent signal. Rho is then activated, binds to Citron-K and translocates it to cell cortex, where the complex is then concentrated in the cleavage furrow by the action of actin cytoskeleton beneath the equator of dividing cells.
Eda
M
M
Department of Pharmacology, Kyoto University Faculty of Medicine, Sakyo, Kyoto 606-8501, Japan.
Yonemura
S
S
Kato
T
T
Watanabe
N
N
Ishizaki
T
T
Madaule
P
P
Narumiya
S
S
D036002:ADP Ribose Transferases / Q000494:pharmacology; D008841:Actin Cytoskeleton / Q000378:metabolism*; D000209:Acute-Phase Proteins / Q000378:metabolism*; D000705:Anaphase / Q000502:physiology; D001905:Botulinum Toxins; D002453:Cell Cycle / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D003593:Cytoplasm / Q000737:chemistry; D049452:Green Fluorescent Proteins; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007399:Interphase / Q000502:physiology; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics; D046911:Macromolecular Substances; D008677:Metaphase / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D008941:Spindle Apparatus / Q000737:chemistry*; D013347:Subcellular Fractions / Q000737:chemistry
D000209:Acute-Phase Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; C010781:acute-phase protein rho; D049452:Green Fluorescent Proteins; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; C115635:citron-kinase; D017346:Protein Serine-Threonine Kinases; D001905:Botulinum Toxins