Rho-dependent transfer of Citron-kinase to the cleavage furrow of dividing cells

Abstract

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.

Inhibiting microcephaly genes as alternative to microtubule targeting agents to treat brain tumors

“…CITK reaches its peak of expression during the G2/M phase of the cell cycle. It is nuclear during interphase, cytoplasmic at early prophase, enriched at spindle poles before anaphase, and is localized at the cleavage furrow and at midbody during cytokinesis [ 115 , 119 ] (Fig. 1 ).…”

Heteroallelic combination of anillin mutants reveals cytoskeletal uncoupling during cytokinesis.

“…It is important to highlight that all these events are coordinated by Rho1-GTP, which is continually generated at the equatorial plasma membrane during cytokinesis (Bement et al, 2005), by the RhoGEF Pebble/ECT2 (Somers and Saint, 2003), and which undergoes GTPase flux (Miller et al, 2008). Both sets of Anillin-scaffolded complexes described in the present study must be controlled by discrete pools of Rho1, since Rho1-GTP-bound Anillin is specifically required for anillo-septin assembly (and thus shedding) (Carim and Hickson, 2023), and Rho1-GTP-bound Citron kinase/Sticky is specifically required for MR formation (Eda et al, 2001; El-Amine et al, 2019; Watanabe et al, 2013). We provide evidence that both of these Rho1-dependent pathways contribute to Anillin localization.…”

Microtubule and Actin Cytoskeletal Dynamics in Male Meiotic Cells of Drosophila melanogaster

“…This study revealed that NMII concentrates at the equatorial cortex and disappears from the poles during anaphase [ 242 ]. The small GTPase RhoA (Rho1 in Drosophila ) is known to control NMII by activating Rho kinase, which, in turn, phosphorylates RLC on Thr-18 and Ser-19 [ 243 , 244 , 245 , 246 ]. It has been proposed that NMII filaments concentrate at the equatorial cortex through a mechanism that requires Rho kinase-mediated RLC phosphorylation.…”