Expression of a phosphodeficient Climp63 mutant results in nuclear fragmentation and prolonged mitosis.

(A) Model of full-length trimeric Climp63 in the ER membrane and its interaction with a microtubule (structure based on PDB 7SJ9). The structure of Climp63 was adapted from an AlphaFold prediction. (B) Multi-sequence alignment of the first 28 amino acids of Climp63 for the indicated species generated by JalView2 (Waterhouse et al., 2009). Experimentally confirmed phosphorylation sites are highlighted by red boxes. Color intensity reflects the degree of conservation. (C) Immunofluorescence analysis of tetracycline-inducible HeLa cell lines expressing Climp63 WT, 3A (S3A, S17A, S19A), or 3E (S3E, S17E, S19E) for 48 h. Sec61β-GFP as an ER marker is stably integrated into these cell lines. White arrows point out mitotic cells that are frequently observed upon 3A expression, yellow arrows point out cells with nuclear fragmentation. Scale bar, 10 µm, N = 3. (D) Representative images of nuclear fragmentation induced by the expression of phosphodeficient Climp63 in HeLa cells and their classification from the experiment in (C). Scale bar, 5 µm. (E) Quantification of the fraction of interphase cells with nuclear fragmentation. N = 3, n ≥ 90, mean ± SEM, *p ≤ 0.05, ns = not significant. (F) Quantification of the percentage of mitotic cells. N = 3, n ≥ 425, mean ± SEM, ****p ≤ 0.0001, ns = not significant. (G) Immunoblot analysis of samples from (C) with the indicated antibodies. N = 3. (H) Quantification of phospho-H3 (pH3) levels in (G) normalized to β-Actin and to the mean within replicates. Asterisks denote significant differences compared to the parental control. Mean ± SD, ***p ≤ 0.001. (I) Immunoblot analysis of phospho-H3 (pH3) levels after Climp63 3A induction with different tetracycline concentrations for 48 h. N = 3. (J) Quantification of protein levels normalized to β-Actin and the uninduced condition from three replicates of (H). Mean ± SD. (K) Representative images of immunofluorescence analysis of HeLa cells in metaphase expressing Climp63 WT, 3A or 3E and stained with the indicated antibodies. Scale bar, 5 µm.

Phosphosite S17A is important to release ER from microtubules in mitosis and is a target of CDK1.

(A) Immunofluorescence analysis of HeLa cell lines expressing Climp63 mutants that are rendered phosphorylation-deficient at individual phosphosites compared to the WT and 3A mutant. Sec61β-GFP as an ER marker is stably integrated into these cell lines. Scale bar, 10 µm. N = 3. (B) Immunoblot analysis of the same samples as in (A) with the indicated antibodies. N = 3. (C) Quantification of the percentage of interphase cells with nuclear fragmentation. N = 3, n ≥ 120, mean ± SEM, *p ≤ 0.05. (D) Quantification of the percentage of mitotic cells. N = 3, n ≥ 420, mean ± SEM. ***p ≤ 0.001. (E) In vitro kinase assay with recombinant purified Cyclin B1-CDK1 and Climp63 1-31-GFP-HA WT or S17E in the presence of γ-[32P]ATP. Histone H1 phosphorylation was used as positive control. N = 3.

The N-terminal 28 amino acids of the cytoplasmic domain of Climp63 are sufficient for MT-ER interactions.

(A) Immunofluorescence analysis of the cytoplasmic domain of Climp63 (Climp63 1-106-HA). Scale bar, 10 µm. N = 3. (B) Schematic depiction of the generated fragments of the cytoplasmic domain of Climp63. All constructs include a transmembrane domain followed by a C-terminal mCherry. The orange line at position 17 indicates the S17A mutation in these constructs and the red line the S17E mutation.(C) Immunofluorescence analysis of tetracycline-inducible HeLa cell lines expressing the depicted Climp63 fragments. Cell lines expressing construct 11-28, 29-132, NDC80 and WALP17 were induced with 1 µg/ml Tetracycline and the other constructs with 20 ng/ml. N = 3. (D) Immunoblot analysis of the same samples as in (C) with the indicated antibodies. N = 3. (E) Quantification of phospho-H3 (pH3) protein levels in (D) normalized to β-Actin and to the mean within replicates. Asterisks denote significant differences compared to the parental control, unless otherwise indicated. Mean ± SD, *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001, N = 4.

Climp63 3A expressing cells arrest in mitosis due to active SAC signaling.

(A) Automated image analysis using CellCognition was used to extract mitotic timing from time-lapse imaging of HeLa cell lines expressing Climp63 WT, 3A or 3E for 24 h and imaged every 3 min for a total of 16 h. Cell trajectories (n ≥ 90) are aligned to the prophase to prometaphase transition and color-coded based on the depicted mitotic phases or cell state. (B) Quantification of prometaphase length from three replicates as in (A). The maximum length is capped off as trajectories are tracked for 150 min after prophase to prometaphase transition, so the full duration of prometaphase in Climp63 3A-expressing cells is unknown. N = 3, n ≥ 400. (C) High resolution time-lapse images of Climp63 WT, 3A or 3E expressing and control HeLa cells progressing through mitosis as in (A). DNA was visualized by SiR-Hoechst and the ER by the stable expression of Sec61β-GFP. Scale bar, 5 µm. (D) Quantification of cells in mitosis upon depletion of MAD2 compared to the control. N = 3, n ≥ 800. (E) Time-lapse images of a representative cell undergoing mitosis upon 20 nM MAD2 siRNA for 48 h or control treatment. Expression of Climp63 3A was induced for 24 h. Scale bar, 5 µm. (F) Representative time-lapse images of mitotic exit in Climp63 3A mutant cells and control upon 320 nM reversine treatment. Reversine was added approximately 10 min before timepoint 0. Scale bar, 5 µm.

Combined time-lapse imaging of tubulin and ER in Climp63 WT and 3A expressing cells.

Representative time-lapse images of Climp63 WT and 3A-expressing HeLa cells progressing through mitosis. DNA was visualized by SPY650-DNA and tubulin by SPY555-tubulin. Scale bar, 5 µm.

Mitotic consequences of persistent MT-ER interactions induced by the expression of phosphodeficient Climp63.

(A) Immunofluorescence analysis of spindle morphology of cells with and without tetracycline-induced expression of Climp63 3A. Images are the maximum projections of z-stacks. Scale bar, 5 µm. (B) Quantification of tubulin morphology from 3D stacks with and without tetracycline-induced expression of Climp63 3A as in (A). Only cells with mostly aligned chromosomes were quantified, as these have likely not been in mitosis for long yet (see Figure 4C). Horizontal line represents the mean. N = 3, n ≥ 18. *p ≤ 0.05. (C) Schematic depiction of NE assembly on a telophase cell. Core components (pink) reassemble in regions with a high MT density, whereas non-core components (green) assemble in regions with low MT density. Lagging chromosomes (represented by the circle) are frequently surrounded by MTs and then assemble only core components, explaining the low NPC density often found on micronuclei formed from lagging chromosomes. (D) Immunofluorescence analysis of fragmented nuclei after 72 h of Climp63 3A expression stained for the indicated antibodies that represent core (Lamin A/C; pink box) and non-core (mAB414; green box) proteins. The white arrow points to a part of the nuclear envelope of a micronucleus that is seemingly missing mAB414 and Lamin A/C signal. The bottom image shows this more clearly with a zoom-in. Upper two scale bars, 10 µm, scale bar of zoom-in, 2 µm, N = 3. (E, F) Representative immunofluorescence images and quantification of the radial distribution of lysosomes (E, α-LAMP1) and mitochondria (F, α-GRP75), which were analyzed in 6 bins using CellProfiler, with and without expression of Climp63 3A. The lines represent the mean intensity per bin, shaded areas ± SD. N = 3, n > 18. Scale bar, 5 µm.