Citron kinase controls a molecular network required for midbody formation in cytokinesis

Abstract

Cytokinesis partitions cytoplasmic and genomic materials at the end of cell division. Failure in this process causes polyploidy, which in turn can generate chromosomal instability, a hallmark of many cancers. Successful cytokinesis requires cooperative interaction between contractile ring and central spindle components, but how this cooperation is established is poorly understood. Here we show that Sticky (Sti), the Drosophila ortholog of the contractile ring component Citron kinase (CIT-K), interacts directly with two kinesins, Nebbish [the fly counterpart of human kinesin family member 14 (KIF14)] and Pavarotti [the Drosophila ortholog of human mitotic kinesin-like protein 1 (MKLP1)], and that in turn these kinesins interact with each other and with another central spindle protein, Fascetto [the fly ortholog of protein regulator of cytokinesis 1 (PRC1)]. Sti recruits Nebbish to the cleavage furrow, and both proteins are required for midbody formation and proper localization of Pavarotti and Fascetto. These functions require Sti kinase activity, indicating that Sti plays both structural and regulatory roles in midbody formation. Finally, we show that CIT-K's role in midbody formation is conserved in human cells. Our findings indicate that CIT-K is likely to act at the top of the midbody-formation hierarchy by connecting and regulating a molecular network of contractile ring components and microtubule-associated proteins.

Fig. 1.

Sti interacts with Neb and Pav via its CC1 region. (A) Schematic diagrams illustrating the protein domains of Sti, Neb, and Pav and the positions of the different fragments used for the in vitro pull-down assays. (B) GST::StiCC1 and GST alone were incubated with in vitro translated and radiolabeled Neb, Pav, and Feo polypeptides (Right) and then pulled down using glutathione beads. (C) The GST::Neb proteins indicated at the top and GST alone were incubated with the in vitro translated and radiolabeled StiCC1, Pav, and Feo polypeptides indicated at the right, and then pulled down using glutathione beads. (D) The GST::Pav proteins indicated at the top and GST alone were incubated with in vitro translated and radiolabeled StiCC1, Neb_1–740, and Feo polypeptides and then pulled down using glutathione beads. (E) GST::StiCC1 and GST alone were incubated with a mixture containing both Neb_741–1121 and Pav_461–685 translated and radiolabeled in vitro and then pulled down using glutathione beads. (F) GST::StiCC1 was incubated with an in vitro translated and radiolabeled Pav_461–685 polypeptide and increasing concentrations of GST::Neb_741–1121. The mixtures were then pulled down using glutathione beads. In all panels, the Ponceau staining of the protein loading is shown in the lower part and the numbers on the left indicate the sizes, in kilodaltons, of the molecular mass marker.

Fig. 2.

Sti colocalizes with Neb at the cleavage furrow, and both proteins partially colocalize with Pav. (A) Drosophila Schneider 2 (S2) cells stably expressing Myc-tagged Sti were fixed and stained to detect Myc, Neb, and tubulin. The dotted lines were used to generate the intensity profiles of the red, green, and blue channels shown in B. (C) Drosophila S2 cells expressing Myc-tagged Sti were fixed and stained to detect Myc, Pav, and tubulin. (D) Drosophila S2 cells stably expressing GFP-tagged Neb_1–1121 (full length), Neb_1–740, Neb_1–118, and Neb_741–1121 were fixed and stained to detect GFP, tubulin (red), and DNA (blue). (E) Drosophila S2 cells stably expressing GFP-tagged full-length Neb were fixed and stained to detect GFP, Pav, and tubulin. (F) Drosophila S2 cells stably expressing GFP-tagged full-length Neb were fixed and stained to detect GFP, Feo, and tubulin. The insets show a 2× magnification of the MB. (Scale bars, 10 µm.)

Fig. 3.

Sti is required for MB formation and proper localization of Pav and Feo. (A–C) Drosophila S2 cells were treated with dsRNAs directed against kanamycin (control) or sti for 72 h and then fixed and stained to detect tubulin, DNA (blue), and Neb, Pav, or Feo. The insets show a 2× magnification of the MB. (Scale bars, 10 µm.) (D) EM images of MBs from cells treated as described earlier. Arrowheads mark the MB matrix. (Scale bars, 1 µm.) (E) Quantification of MB defects from the experiments shown in A–C. More than 200 mid-late telophase cells were counted in each experiment (n = 4). Bars indicate SEs. *P < 0.05 (Mann–Whitney U test). (F) Drosophila S2 cells stably expressing Myc, StiWT::Myc, or StiKD::Myc were treated with dsRNAs directed against either kanamycin (control) or the 3′ UTR of sti for 72 h. The number of cells showing MB defects was then counted and plotted. Only Myc-positive cells were counted, and more than 200 mid-late telophase cells were counted in each experiment (n = 4). Bars indicate SEs. *P < 0.05 (Mann–Whitney U test). Note that sti 3′UTR RNAi is less efficient than RNAi targeting Sti ORF (17).

Fig. 4.

Neb depletion phenocopies sti RNAi. (A–C) Drosophila S2 cells were treated with dsRNAs directed against kanamycin (control) or neb for 96 h and then fixed and stained to detect tubulin, DNA (blue), and Sti, Pav, or Feo. The insets show a 2× magnification of the MB. (Scale bars, 10 µm.) (D) EM images of MBs from cells treated as described earlier. Arrowheads mark the MB matrix. Note in the right panel that the MB matrix is mispositioned inside the cell body. (Scale bars, 1 µm.) (E) Quantification of multinucleate cells and MB defects from the experiments shown in A–C. More than 600 and 200 cells were counted (n = 4) for the quantification of multinucleate cells and central spindle defects, respectively. Bars indicate SEs. *P < 0.05 (Mann–Whitney U test).

Fig. 5.

CIT-K is necessary for proper MB formation in HeLa cells. (A) In vitro pull-down assay using the GST::MKLP1 proteins indicated at the top or GST alone and in vitro translated and radiolabeled CIT-K CC1 fragment. (B) HeLa cells were treated with siRNAs directed against scrambled sequences (control) or CIT-K for 48 h and then fixed and stained to detect tubulin, DNA (blue), and either MKLP1 or PRC1. The insets show a 2.5× magnification of the MB. (Scale bars, 20 µm.) (C) Quantification of MB defects and multinucleate cells from the experiments shown in B. More than 120 cells were counted in each experiment (n = 4). Bars indicate SEs. *P < 0.05 (Mann–Whitney U test).

Fig. 6.

Model depicting the role of Sti in MB formation. Cartoon illustrating Sti interactions during MB formation. Anillin and Rho1 are not included.