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, 195 (4), 595-603

Sticky/Citron Kinase Maintains Proper RhoA Localization at the Cleavage Site During Cytokinesis

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Sticky/Citron Kinase Maintains Proper RhoA Localization at the Cleavage Site During Cytokinesis

Zuni I Bassi et al. J Cell Biol.

Abstract

In many organisms, the small guanosine triphosphatase RhoA controls assembly and contraction of the actomyosin ring during cytokinesis by activating different effectors. Although the role of some RhoA effectors like formins and Rho kinase is reasonably understood, the functions of another putative effector, Citron kinase (CIT-K), are still debated. In this paper, we show that, contrary to previous models, the Drosophila melanogaster CIT-K orthologue Sticky (Sti) does not require interaction with RhoA to localize to the cleavage site. Instead, RhoA fails to form a compact ring in late cytokinesis after Sti depletion, and this function requires Sti kinase activity. Moreover, we found that the Sti Citron-Nik1 homology domain interacts with RhoA regardless of its status, indicating that Sti is not a canonical RhoA effector. Finally, Sti depletion caused an increase of phosphorylated myosin regulatory light chain at the cleavage site in late cytokinesis. We propose that Sti/CIT-K maintains correct RhoA localization at the cleavage site, which is necessary for proper RhoA activity and contractile ring dynamics.

Figures

Figure 1.
Figure 1.
CR dynamics in sti mutant neuroblasts. (A and B) Kymographs of time-lapse imaging of sqh-GFP in a control stiGS9053/+ heterozygous (A) or stiGS9053/Df(3L)iro2 hemizygous mutant (B) dividing larval brain neuroblast showing the progression of cytokinesis every 30 s. Bar, 1 µm. (C and D) Equatorial cell diameter during cytokinesis measured during live imaging of control stiGS9053/+ (C; n = 15) or stiGS9053/Df(3L)iro2 mutant (D; n = 14) neuroblast divisions. Time is indicated in seconds after Sqh-GFP concentration at the cell equator. All cells imaged completed contraction within 5 min, with sti mutants showing both faster and slower contractions than the more uniform controls.
Figure 2.
Figure 2.
Sti localization to the CF depends on RhoA and CR assembly. (A) Drosophila S2 cells were treated with dsRNAs directed against the bacterial kanamycin resistance gene (absent in Drosophila and used as a control) or Rho1 for 72 h and then fixed and stained to detect Sti, tubulin, and DNA. (B) S2 cells were treated with dsRNAs directed against kanamycin (control) or zipper for 84 h and then fixed and stained to detect Sti, tubulin, and DNA. Percentages on the left indicate localization of Sti. (C) S2 cells were treated with dsRNAs directed against kanamycin (control) or zipper for 84 h and then fixed and stained to detect Rho1, Sti, tubulin, and DNA (not depicted in the merged images). (D) Drosophila S2 cells were treated with latrunculin A (Lat-A) or its solvent DMSO for 1 h and then fixed and stained to detect Rho1, Sti, tubulin, and DNA (not depicted in the merged images). Bars, 10 µm.
Figure 3.
Figure 3.
Sti localizes to the CF via interaction of its CC region with actin and myosin. (A) Schematic representation illustrating Sti protein domains and the localization of different Sti fragments tagged with GFP. (B) Drosophila S2 cells stably expressing various GFP-tagged Sti fragments (indicated at the top) were fixed and stained to detect GFP, tubulin, and DNA (blue). The arrow in the StiFL image marks a midbody remnant. In the CC1 image, the arrowhead marks the spindle midzone, whereas the arrow indicates CC1 localization to centrosome and astral microtubules. GFP::CC2a localization is shown in early and late telophase and after treatment for 72 h with a dsRNA directed against the CNH region (sti RNAi). Bars, 10 µm. (C) S2 cells stably expressing either the Sti CC2a fragment tagged with PtA or PtA alone were transfected with a Myc::Sqh construct for 48 h, and then protein extracts were used in a PtA pull-down assay. The extracts and pull-downs were analyzed by Western blotting to detect Myc::Sqh, actin, Rho1, and PtA. (D) S2 cells stably expressing PtA::StiCC2a were transfected with a Myc::Sqh construct for 48 h and, 5 h before collection, treated either with Cytochalasin D (Cyto D) or its solvent DMSO. Protein extracts were then subject to PtA pull-down assay, and extracts and pull-downs were analyzed by Western blotting to detect Myc::Sqh, actin, Rho1, and PtA. Numbers on the left indicate the sizes in kilodaltons of the molecular mass markers.
Figure 4.
Figure 4.
Sti is required for proper localization of Rho1 at the CF. (A) Drosophila S2 cells were treated with dsRNAs directed against kanamycin (control) or sti for 72 h and then fixed and stained to detect Rho1, tubulin, and DNA (blue). (B) GST::Rho1 variants were preincubated with GTP and then mixed with Sti1,150–1,331, Sti1,437–1,854, or Rhotekin RBD, translated, and radiolabeled in vitro. The mixtures were then pulled down using glutathione beads, separated on SDS-PAGE gels, transferred onto nitrocellulose membrane, and exposed to x-ray films. The Ponceau S staining of protein loading is shown at the bottom. The numbers on the left indicate the sizes in kilodaltons of the molecular mass marker. (C) S2 cells stably expressing Myc alone or Myc-tagged Sti full length (StiFL), StiΔCNH, or a kinase-dead version of Sti (StiKD) were treated with dsRNAs directed against either kanamycin (kana) or the 3′ UTR of sti for 72 h. The number of multinucleate cells was then counted and plotted. Only Myc-positive cells were counted, and >600 cells were counted in each experiment. (D) Quantification of the cells showing aberrant Rho1 localization from experiments performed as described in C. At least 120 cells were counted in each experiment. (E) Cells expressing the transgenes described in C and depleted of endogenous Sti were stained to detect Rho1, Myc, and tubulin. Bars, 10 µm. n = 4; *, P < 0.05 (Mann–Whitney U test). Error bars indicate standard deviations.
Figure 5.
Figure 5.
Sti depletion causes an increase of phosphorylated Sqh at the CF in late cytokinesis. (A) Drosophila S2 cells stably expressing Sqh::GFP were treated with dsRNAs directed against kanamycin (control) or sti for 72 h and then fixed and stained to detect Sqh::GFP, tubulin, and DNA (blue). The box plots showing the quantification of Sqh::GFP fluorescence levels at the CF in early and late cytokinesis are shown at the bottom. The intensity of Sqh::GFP fluorescence at the CF was calculated using the formula shown, in which ICF is the fluorescence intensity at the CF, and IC represents the background intensity measured within an identical area inside the cytoplasm. At least 30 cells from two separate experiments were analyzed. AU, arbitrary unit. (B) S2 cells were treated with dsRNAs directed against the kanamycin gene (control), sti, or rok for 72 h and then fixed and stained to detect Sqh1P, tubulin, and DNA (blue). Quantification of Sqh1P levels was performed as described in A. Note that the Sqh1P staining on centrosomes is not specific because it was not affected by ROK depletion, and Sqh::GFP never localized to centrosomes (Fig. S2 B). **, P < 0.01 (Student’s t test). (C) S2 cells were treated with dsRNAs directed against kanamycin (control), sti, or rok for 72 h and then fixed and stained to detect Sqh2P, tubulin, and DNA (blue). In each box plot, the box contains the values comprised between the 25th and 75th percentile, and the horizontal line inside the box marks the median. The ends of the whiskers indicate the minimum and maximum values. Bars, 10 µm.

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