Persistent contacts between Climp63 and microtubules cause mitotic defects and nuclear fragmentation

Reviewer #1 (Public review):

Summary:

In the present manuscript, de Bos and Kutay investigate the functional implications of persistent microtubule-ER contacts as cells go through mitosis. To do so, they resorted to investigating phosphorylation mutants of the ER-Microtubule crosslinker Climp63. They found that phosphodeficient Climp63 mutants induce a severe SAC-dependent mitotic delay after normal chromosome alignment, with an impressive mitotic index of approximately 75%. Strikingly, this was often associated with massive nuclear fragmentation into up to 30 micronuclei that are able to recruit both core and non-core nuclear envelope components. One particular residue (S17) that is phosphorylated by Cdk1 seems to account for most, if not all, these phenotypes. Furthermore, the authors use the impact on mitosis as an indirect way to map the microtubule binding domain of Climp63, which has remained controversial, and found that it is mostly restricted to the N-terminal 28 residues of Climp63. Of note, despite the strong impact on mitosis, persistent microtubule-ER contacts did not affect the distribution of other organelles during mitosis, such as mitochondria or lysosomes.

Strengths:

Overall, this work provides important mechanistic insight into the functional implications of ER-microtubule network remodelling during mitosis and should be of great interest to a vast readership of cell biologists.

Weaknesses:

Some of the key findings appear somewhat preliminary and would be worth exploring further to substantiate some of the claims and clarify the respective impact on mitosis and nuclear envelope reassembly on the resulting micronuclei.

The following suggestions would significantly clarify some key points:

(1) The striking increase in mitotic index in cells expressing the Climp63 phosphodefective mutant, together with their live cell imaging data indicating extensive mitotic delays that can be relieved by SAC inhibition, suggests that SAC silencing is significantly delayed or even impossible to achieve. The fact that most chromosomes align in 12 min, irrespective of the expression of the Climp63 phosphodefective mutant, suggests that initial microtubule-kinetochore interactions are not compromised, but maybe cannot be stably maintained. Alternatively, the stripping of SAC proteins from kinetochores by dynein along attached microtubules might be compromised, despite normal microtubule-kinetochore attachments. The authors allude to both these possibilities, but unfortunately, they never really test them. This could easily be done by immunofluorescence with a Mad1 or c-Mad2 antibody to inspect which fraction of kinetochores (co-stained with a constitutive kinetochore marker, such as CENP-A or CENP-C) are positive for these SAC proteins. If just a small fraction, then the stability of some attachments is likely the cause. If most/all kinetochores retain Mad1/c-Mad2, then it is probably an issue of silencing the SAC.

(2) The authors use the increase in mitotic index (H3 S10 phosphorylation levels) as a readout for the MT binding efficiency of Climp63 and respective mutants. Although suggestive, this is fairly indirect and requires additional confirmation. For example, the authors could perform basic immunofluorescence in fixed cells to inspect co-localization of Climp63 (and its mutants) with microtubules.

(3) The authors refer in the discussion that the striking nuclear fragmentation seen upon mitotic exit of cells expressing Climp63 phosphodefective mutant has not been reported before, and yet it is strikingly similar to what has been previously observed in cells treated with taxol (they cite Samwer et al. 2017, but they might elect to cite also Mitchison et al., Open Biol, 2017 and most relevantly Jordan et al., Cancer Res, 1996). This striking similarity and given the extensive mitotic delay observed in the Climp63 phosphodefective mutant, it is tempting to speculate that these cells are undergoing mitotic slippage (i.e., cells exit mitosis without ever satisfying the SAC) because they are unable to silence/satisfy the SAC. Indeed, the scattered micronuclei morphology has also been observed in cells undergoing mitotic slippage (e.g., Brito and Rieder, Curr Biol., 2006). The experiment suggested in point #1 should also shed light on this problem. The authors might want to consider discussing this possible explanation to interpret the observed phenotypes.

(4) One of the most significant implications of the findings reported in this paper is that microtubule proximity does not seem to impact the assembly of either core or non-core nuclear envelope proteins on micronuclei (that possibly form due to mitotic slippage, rather than normal anaphase). These results challenge some models explaining nuclear envelope defects in micronuclei derived from lagging chromosomes due to the proximity of microtubules, and, as the authors point out at the very end, other reasons might underlie these defects. Along this line, the authors might elect to cite Afonso et al. Science, 2014, and Orr et al., Cell Reports, 2022, who provide evidence that a spindle midzone-based Aurora B gradient, rather than microtubules per se, underlie the nuclear envelope defects commonly seen in micronuclei derived from lagging chromosomes during anaphase.

Reviewer #2 (Public review):

Mitotic phosphorylation of the ER-microtubule linker CLIMP63 was discovered decades ago and was shown to release CLIMP63 from microtubules. Here, the authors describe for the first time the significance of CLIMP63 phosphorylation for mitotic division in cells. Expression of non-phosphorylatable CLIMP63 led to a massive re-localization of ER into the area of the mitotic spindle. This was not unexpected, as another ER-microtubule linker, STIM1, is phosphorylated during mitosis to release it from microtubules, and unphosphorylatable STIM1 also leads to an invasion of the ER into the spindle. The authors map CLIMP63's microtubule-binding domain and define S17 as the critical residue that needs to be phosphorylated for release from microtubules and as a target of Cdk1, albeit with an indirect assay that is based on the ability of overexpressed mutants to disrupt mitosis. The authors further demonstrate that aberrant, microtubule-tethered membranes in the spindle disrupt spindle function. This is in line with the group's prior findings that chromosome-tethered membranes lead to severe chromosome segregation defects. Cells overexpressing phospho-deficient CLIMP63 arrested in prometaphase with an active checkpoint. When these cells were forced to exit mitosis, a large number of micronuclei formed. Interestingly, these micronuclei had different compositions and properties from previously described ones, suggesting that there are diverse paths for a cell to become multinucleated. Lastly, the authors asked whether mitochondria and lysosomes depend on ER for their distribution in mitotic cells. However, the position of these other organelles was unchanged in cells in which ER was re-localized due to the overexpression of phospho-deficient CLIMP63. This is an interesting observation in the context of how the interior organisation of mitotic cells is achieved.

Suggestions:

(1) The authors should confirm the mapping of the microtubule-binding domain by more direct assays, such as microtubule co-pelleting or proximity ligation assays.

(2) The authors should clarify why they performed phenotypic studies and live microscopy experiments (Figures 4 and 5) using the CLIMP63(3A) mutant, despite knowing that the relevant phosphorylation site was S17. Were the phenotypes different for S17A versus the triple mutant?