NudCL2 is required for cytokinesis by stabilizing RCC2 with Hsp90 at the midbody

Abstract

Cytokinesis is required for faithful division of cytoplasmic components and duplicated nuclei into two daughter cells. Midbody, a protein-dense organelle that forms at the intercellular bridge, is indispensable for successful cytokinesis. However, the regulatory mechanism of cytokinesis at the midbody still remains elusive. Here, we unveil a critical role for NudC-like protein 2 (NudCL2), a co-chaperone of heat shock protein 90 (Hsp90), in cytokinesis regulation by stabilizing regulator of chromosome condensation 2 (RCC2) at the midbody in mammalian cells. NudCL2 localizes at the midbody, and its downregulation results in cytokinesis failure, multinucleation, and midbody disorganization. Using iTRAQ-based quantitative proteomic analysis, we find that RCC2 levels are decreased in NudCL2 knockout (KO) cells. Moreover, Hsp90 forms a complex with NudCL2 to stabilize RCC2, which is essential for cytokinesis. RCC2 depletion mirrors phenotypes observed in NudCL2-downregulated cells. Importantly, ectopic expression of RCC2 rescues the cytokinesis defects induced by NudCL2 deletion, but not vice versa. Together, our data reveal the significance of the NudCL2/Hsp90/RCC2 pathway in cytokinesis at the midbody.

Keywords: Hsp90; NudCL2; RCC2; cytokinesis; midbody.

Figure 1.

Downregulation of NudCL2 causes cytokinesis failure. (A) Schematic representation of NudCL2 gene targeting strategy. (B) Western blot analysis of NudCL2 protein in entire WT and NudCL2 KO HEK-293 cells. (C) Time-lapse DIC images of the live cell imaging experiment of the control or NudCL2 KO cells. Time point 00:00 (hours:min) refers to the first frame where the separating sister chromatids are observed. (D) Percentage of WT (n = 189), KO-1 (n = 157) or KO-2 (n = 155) cells showing cytokinesis failure was calculated. (E) Cells were stained with DAPI and anti-α-tubulin antibody for immunofluorescence analysis. The percentage of WT (n = 905), KO-1 (n = 1264), or KO-2 (n = 1093) cells in each mitotic phase and cytokinesis was calculated. (F) Cells transfected with Myc or Myc-NudCL2 vector were subjected to Western blot with the antibodies as shown. (G) The percentage of transfected cells in telophase and cytokinesis was calculated using immunofluorescence analysis. The number of calculated cells in WT + Myc, KO-1 + Myc, KO-2 + Myc, WT + Myc-NudCL2, KO-1 + Myc-NudCL2 or KO-2 + Myc-NudCL2 was 1,590, 729, 839, 1,717, 1,101, or 747. One-way ANOVA with multiple comparison corrections was performed. (H) Cells were stained with DAPI and anti-α-tubulin antibody for immunofluorescence. White arrowheads indicate the multinucleated cells. (I) The percentages of the multinucleated cells in WT (n = 841), KO-1 (n = 1,135), or KO-2 (n = 1,009) were calculated. (J) Cells transfected with the indicated vectors were stained to detect DNA and α-tubulin. The percentages of the multinucleated cells (from left to right: n = 1,297, 795, 676, 1,429, 952, and 664) were calculated. One-way ANOVA with multiple comparison corrections was performed. (K and L) Schematic illustration of the rapid downregulation strategy of NudCL2 using the AID system. OsTIR1, the Oryza sativa TIR1; T2A, thosea asigna virus 2A; mAID, the degron termed mini-AID. Expressed OsTIR1 binds to the SCF E3 ubiquitin ligase complex (including Skp1, Cul1, and Rbx1) to form a functional complex with the endogenous components in cells. When Auxin interacts with OsTIR1 and mAID, SCF-OsTIR1 recruits an E2 ligase to induce polyubiquitylation and degradation of NudCL2-mAID protein. (M) The workflow of NudCL2 downregulation by AID system in cells stably expressing OsTIR1 and NudCL2-mAID using NudCL2 KO cell (NudCL2-mAID cells). (N) The NudCL2-mAID cells were synchronized into anaphase by thymidine-nocodazole block and release procedure, and treated with or without auxin. Then the entire cells were harvested immediately at different time points, and subjected to Western blot with anti-mAID antibody. (O and P) The synchronized NudCL2-mAID cells were subjected to live cell imaging experiments. The first frame of time-lapse DIC images was taken at 15 min after auxin and Hoechst 33342 addition and the time point was marked as 00:15 (hours:min). Percentage of mock (n = 131) or auxin-treated (n = 112) cells showing cytokinesis failure was calculated. β-Actin, a loading control. Scale bars, 5 μm. Higher magnifications of the boxed regions are displayed. Quantitative data are expressed as the mean ± SD (from three biological replicates). The P values were calculated using Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, no significance.

Figure 2.

Loss of NudCL2 disrupts the midbody organization. (A–F) Cells were fixed and subjected to immunofluorescence analyses with the antibodies as shown. (G) HEK-293 cells were synchronized with a thymidine-nocodazole block, released, and harvested. The proteins from the total protein extracts (Tot extracts) and midbodies were extracted and subjected to Western blot analysis with the indicated antibodies. (H) Control and NudCL2 KO cells were fixed and subjected to immunofluorescence analysis with anti-Aurora B and anti-α-tubulin antibodies. (I) Intensity line scan plots for corresponding proteins of images. The frequencies of WT (n = 166), KO-1 (n = 168), or KO-2 (n = 166) cells with mislocalization of Aurora B at the midbody were calculated. (J) Control and NudCL2 KO cells were fixed and subjected to immunofluorescence analysis with anti-MKLP1 and anti-PLK1 antibodies. (K) Intensity line scan plots for corresponding proteins of images. The frequencies of WT (n = 158), KO-1 (n = 158), or KO-2 (n = 157) cells with mislocalization of MKLP1 (K) or PLK1 (L) at the midbody were calculated. DNA was visualized with DAPI. Scale bars, 5 μm. Higher magnifications of the boxed regions are displayed. Intensity of line scan plots was quantified and graphed using ImageJ software. White arrows indicate the direction of line scan plots. Quantitative data are expressed as the mean ± SD (from three biological replicates). The P values were calculated using Student’s t-test. **P < 0.01, ***P < 0.001; ns, no significance.

Figure 3.

Loss of NudCL2 decreases the RCC2 protein levels in the midbody. (A) Schematic representation of the isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analysis. (B and C) Volcano plot showing P values (−log₁₀) versus the protein ratio of KO-1/WT cells (log₂) or KO-2/WT cells (log₂). Proteins exhibiting a fold change > 1.2 or < 0.83, P < 0.05 were defined as “differentially expressed proteins” (red dots). Others were defined as “no change” (blue dots). (D) Scatter plot showing the protein ratio of KO-1/WT cells (log₂) versus KO-2/WT cells (log₂). Each point represents a single protein identified. (E) Venn diagram showing the overlap among the proteins downregulated in KO-1 and KO-2 of the quantitative proteomic analysis and the midbody proteins from the MiCroKiTS database. (F) Two midbody proteins that overlap in Fig. 3E are shown. (G) Western blot analysis of proteins from entire control and NudCL2 KO cells using the indicated antibodies. (H) Western blot analysis of proteins from entire control and NudCL2 KO cells transfected with Myc or Myc-NudCL2 vector with the indicated antibodies. (I) The qRT-PCR analysis of RCC2 mRNA in control and NudCL2 KO cells. ns, no significance, Student’s t-test. (J) Western blot analysis of total protein extracts (Tot extracts) and midbodies purified from control and NudCL2 KO cells with the indicated antibodies. (K) Control and NudCL2 KO cells were fixed and subjected to smFISH to detect RCC2 mRNA, followed by immunofluorescence analysis with the anti-RCC2 antibody. (L) The qRT-PCR analysis of RCC2 mRNA in total extracts and midbodies purified from synchronized telophase control and NudCL2 KO HEK-293 cells. ns, no significance, Student’s t-test. (M) Cells were synchronized with a thymidine-nocodazole block/release and labeled with puromycin with or without CHX. Then, the midbodies were purified and subjected to immunoprecipitation analysis with anti-RCC2 antibody. (N) Western blot analysis of proteins from entire control and NudCL2 KO cells treated with 100 μg/mL CHX using the indicated antibodies. The intensity of each band was measured using ImageJ software. The relative amounts of RCC2 were calculated after normalization (RCC2/β-actin). ****P < 0.0001, two-way ANOVA. (O) Western blot analysis of protein from entire cells treated with 1 μmol/L MG132 for 24 h. (P) The entire cells were transfected with the indicated vectors and immunoprecipitation analysis was performed using anti-Flag antibody. (Q) Immunoprecipitation analysis was performed using the indicated antibodies. (R) In vitro GST pull-down assays using purified GST, GST-NudCL2, and His-RCC2 protein. (S) Cells were subjected to immunofluorescence analyses with the indicated antibodies. DNA was visualized with DAPI. Higher magnifications of the boxed regions are displayed below. Scale bars, 5 μm. β-Actin, a loading control. 5% of the total input is shown.

Figure 4.

RCC2 interacts with NudCL2 and Hsp90. (A–C) Immunoprecipitation analysis was performed using the indicated antibodies. (D) In vitro GST pull-down assays using purified GST-NudCL2 with His-RCC2 and His-Hsp90 proteins. (E) Cells were subjected to immunofluorescence analysis with the indicated antibodies. (F) Cells transfected with Myc-Hsp90 vector were subjected to immunofluorescence analysis with the indicated antibodies. (G) Cells were subjected to immunofluorescence analyses with the indicated antibodies. (H) Western blot analysis of total protein extracts (Tot extracts) and midbodies purified from telophase cells with the indicated antibodies. (I) Cells were subjected to immunofluorescence analyses with the indicated antibodies. DNA was visualized with DAPI. Scale bars, 5 μm. Higher magnifications of the boxed regions are shown. 5% of the total input is shown.

Figure 5.

Hsp90 is involved in cytokinesis by regulating RCC2 with NudCL2. (A and B) The entire cells treated with different concentrations of GA for 48 h (A) or 0.5 μmol/L GA at the indicated time points (B) were subjected to Western blot analyses with the indicated antibodies. (C) The entire cells were treated with 0.5 μmol/L GA for 48 h and subjected to qRT-PCR analysis. (D and E) Cells were treated with 0.5 μmol/L GA. After 24 h, cells were added with 100 μg/mL CHX and harvested at the indicated times, then subjected to Western blot analysis using the indicated antibodies. (D) Cells were subjected to immunofluorescence analysis with the indicated antibodies. (E and F) The entire cells were treated with 0.5 μmol/L GA. After 24 h, cells were added with 100 μg/mL CHX and harvested at the indicated times, then subjected to Western blot analysis using the indicated antibodies (E). The intensity of each band was measured using ImageJ software and the relative protein levels of RCC2 were normalized to β-actin (F). Quantitative data are shown from three biological replicates. All values are expressed as mean ± SEM; statistical analyses were performed using two-way ANOVA. (G) The entire cells were treated with 0.5 μmol/L GA for 24 h, then incubated with 1 μmol/L MG132 for another 24 h and subjected to Western blot analysis. β-Actin, a loading control. (H) Cells were stained with DAPI and anti-α-tubulin antibody. The percentages of the multinucleation in DMSO (n = 2,218) and GA-treated (n = 1,469) cells were calculated. (I–M) Cells were subjected to an immunofluorescence experiment as described in Fig. 2H–K. The frequencies of DMSO or GA-treated cells with Aurora B (n = 292 or 206), MKLP (n = 209 or 220) or PLK1 (n = 148 or 162) mislocalization were calculated. (N and O) Cells transfected with Myc or Myc-Hsp90 were subjected to Western blot and immunofluorescence analyses as described in Fig. 1H. The percentages of the multinucleation (from left to right: n = 1,662, 1,736, 1,102, and 1,797) were calculated. (P and Q) Cells treated with or without GA with the indicated vectors were subjected to Western blot and immunofluorescence analyses as described in Fig. 1H. The percentages of the multinucleation (from left to right: n = 3,785, 2,912, 4,937, and 4,195) were calculated. DNA was visualized with DAPI. Scale bars, 5 μm. Higher magnifications of the boxed regions are shown. β-Actin, a loading control. Quantitative data are expressed as the mean ± SD from the three biological replicates. The P values were calculated using Student’s t-test. ***P < 0.001, ****P < 0.0001; ns, no significance.

Figure 6.

RCC2 participates in cytokinesis as the downstream target of NudCL2. (A) HEK-293 cells transfected with control or RCC2 siRNAs were subjected to Western blot analysis from the entire cells. (B and C) Time-lapse DIC images of the live cell imaging experiment of cells transfected with RCC2 siRNAs. Percentage of siCtrl (n = 186), siRCC2-1 (n = 101) or siRCC2-2 (n = 75) cells showing cytokinesis failure was calculated. (D) The transfected cells with the siRNAs as shown were fixed and stained to detect DNA and α-tubulin. The percentages of the multinucleation in siCtrl (n = 1,069), siRCC2-1 (n = 1,117) and siRCC2-2 (n = 1,135) cells were calculated. (E–I) HEK-293 cells transfected with the indicated siRNAs were fixed and subjected to immunofluorescence analyses with anti-α-tubulin and anti-Aurora B (E), anti-MKLP1, and anti-PLK1 (G) antibodies. The frequencies of siCtrl, siRCC2-1 or siRCC2-2 cells with Aurora B (n = 161, 162, or 161), MKLP1 (n = 160, 161, or 159) or PLK1 (n = 157, 158, or 157) mislocalization were calculated. (J) Schematic illustration of the rapid downregulation strategy of RCC2 using the AID system. (K) The RCC2 KO cells expressing OsTIR1-T2A-RCC2-mAID were synchronized into anaphase by thymidine-nocodazole blocking and releasing for 30 min. Then cells were treated with or without auxin and harvested immediately at different time points, and subjected to Western blot with anti-mAID antibodies from the entire cells. (L and M) Time-lapse DIC images of the live cell imaging experiment of the RCC2 KO cells expressing OsTIR1-T2A-RCC2-mAID. Cells were synchronized into anaphase by thymidine-nocodazole blocking and  releasing  for 30 min, then treated with auxin and Hoechst 33342, after 15 min, subjected to the live cell imaging experiment. The first frame of images was taken at 15 min after auxin addition and the time point was marked as 00:15 (hours:min). Percentage of mock (n = 142) or auxin-treated (n = 134) cells showing cytokinesis failure was calculated. (N and O) Control and NudCL2 KO cells were transfected with Myc or Myc-RCC2 vector and subjected to Western blot from the entire cells and immunofluorescence analyses using the indicated antibodies. The percentages of the multinucleated cells (from left to right: n = 770, 923, 708, and 566) were calculated. (P and Q) Control or RCC2 siRNA cells transfected with Myc or Myc-NudCL2 vector were subjected to Western blot from the entire cells and immunofluorescence analyses using the indicated antibodies. The percentages of the multinucleated cells (from left to right: n = 2,007, 3,011, 1,505, and 2,403) were calculated. (R) Working model for the role of NudCL2 in cytokinesis. NudCL2, Hsp90, the mRNA, and the protein of RCC2 all localize at the midbody arm. NudCL2 collaborates with Hsp90 to stabilize RCC2 protein and ensure successful abscission, while loss of NudCL2 decreases RCC2 at the midbody to result in cleavage furrow regression and multinucleation. β-Actin, a loading control. Scale bars, 5 μm. Quantitative data are expressed as the mean ± SD (from three biological replicates). The P values were calculated using Student’s t-test. **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, no significance.