Contractile peri-nuclear actomyosin network repositions peripheral and polar chromosomes to promote early kinetochore–microtubule interactions

  1. Nooshin Sheidaei
  2. John K. Eykelenboom
  3. Zuojun Yue
  4. Graeme Ball
  5. Alexander J.R. Booth
  6. Tomoyuki U. Tanaka

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Abstract

For correct segregation of chromosomes in mitosis, they must efficiently and properly interact with the mitotic spindle during prometaphase. For this, the locations of chromosomes in the nucleus or relative to spindle poles are crucial – chromosomes at the nuclear periphery or behind spindle poles (polar regions) interact less efficiently with the mitotic spindle and show higher risks of missegregation in the subsequent anaphase. Nonetheless, the missegregation rate of such chromosomes is still relatively low in unperturbed normal cells. Thus, unknown mechanisms may mitigate the risks of their missegregation. We previously found that the actomyosin network (PANEM) is formed on the cytoplasmic side of the nuclear envelope during prophase, and its myosin-II-dependent contraction facilitates chromosome interaction with the mitotic spindle, shortly after nuclear envelope breakdown (NEBD). However, it remains unclear which chromosome interaction steps are facilitated or which chromosomes specifically benefit from it. Here, we show that the PANEM contraction directly pushes chromosomes located at the nuclear periphery inward immediately after NEBD. Tracking motions of individual kinetochores reveals that the PANEM contraction facilitates kinetochores’ initial interaction with spindle microtubules, but does not affect their subsequent poleward motion. The PANEM contraction also promotes the onset of their congression towards the spindle mid-plane, but does not affect congression itself once it starts. Furthermore, the PANEM contraction reduces the volume of polar regions, and helps reposition chromosomes from these regions and initiate their congression. Impaired PANEM contraction results in defective chromosome congression and frequent chromosome missegregation. In conclusion, shortly after NEBD, the PANEM contraction repositions chromosomes from unfavorable locations, i.e. the nuclear periphery and polar regions, to facilitate productive kinetochore-microtubule interaction and ensure high-fidelity chromosome segregation.

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    Referee #3

    Evidence, reproducibility and clarity

    Sheidaei et al., report how chromosomes are brought to positions that facilitate kinetochore-microtubule interactions during mitosis. The study focusses on an important early step of the highly orchestrated chromosome segregation process. Studying kinetochore capture during early prophase is extremely difficult due to kinetochore crowding but the team has taken up the challenge by classifying the types of kinetochore movements, carefully marking kinetochore positions in early mitosis and linking these to map their fate/next-positions over time. The work is an excellent addition to the field as most of the literature has thus far focussed on tracking kinetochore in slightly later stages of mitosis. The authors show that the PANEM facilitates chromosome positioning towards the interior of the newly forming spindle, which in turn facilitates chromosome congression - in the absence of PANEM chromosomes end up in unfavourable locations, and they fail to form proper kinetochore-microtubule interactions. The work highlights the perinuclear actomyosin network in early mitosis (PANEM) as a key spatial and temporal element of chromosome congression which precedes the segregation process.

    Major points

    1. The complexity of tracking has been managed by classifying kinetochore movements into 4 categories, considering motions towards or away from the spindle mid-plane. While this is a very creative solution in most cases, there may be some difficult phases that involve movement in both directions or no dominant direction (eg Phase3-like). It is unclear if all kinetochores go through phase1, 2, 3 and 4 in a sequential or a few deviate from this pattern. A comment on this would be helpful. Also, it may be interesting to compare those that deviate from the sequence, and ask how they recover in the presence and absence of azBB.

    2. Would peripheral kinetochore close to poles behave differently compared to peripheral kinetochore close to the midplane (figure S4) ?In figure 3D, are they separated? If not, would it look different?

    3. Uncongressed polar chromosomes (eg., CENPE inhibited cells) are known to promote tumbling of the spindle. In figure 5B with polar chromosomes, it will be helpful to indicate how the authors decouple spindle pole movements from individual kinetochore movements.

    4. The work has high quality manual tracking of objects in early mitosis- if this would be made available to the field, it can help build AI models for tracking. The authors could consider depositing the tracking data and increasing the impact of their work.

    Minor points

    1. It will be helpful for readers to see how many kinetochores/cell were considered in the tracking studies. Figure legends show kinetochore numbers but not cell numbers.
    2. Discussion point: If cells had not separated their centrosomes before NEBD, would PANEM still be effective? Perhaps the cancer cell lines or examples as shown in Figure 6A have some clues here.
    3. Figure 7 cartoon shows misalignment leading to missegregation. It may be useful to consider this in the context of the centrosome directed kinetochore movements via pivoting microtubules. Is this process blocked in azBB treated cells?
    4. Are all the N-CIN- lines with PANEM highly sensitive to azBB? In other words, is PANEM essential for normal congression in some of these lines.
    5. Are congression times delayed in lines that naturally lack PANEM?
    6. Page 23 "we first identified the end of congression" how does this relate to kinetochore oscillations that move kinetochores away from the metaphase plate?
    7. Are spindle pole distances (spindle sizes) different in early and late mitotic cells (4min vs 6min after NEBD) in control vs azBB treated cells? Please comment on Figure S2E (mean distance) in the context of when phase 4 is completed. Does spindle size return to normal after congression?

    Significance

    The current work builds upon their previous work, in which the authors demonstrated that an actomyosin network forms on the cytoplasmic side of the nuclear envelope during prophase. This work explains how the network facilitates chromosome capture and congression by tracking motions of individual kinetochores during early mitosis. The findings can be broadly useful for cell division and the cytoskeletal fields.

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, Sheidaei et al. reported on their study of chromosome congression during the early stages of mitotic spindle assembly. Building on their previous study (ref. #15, Booth et al., Elife, 2019), they focused on the exact role of the actin-myosin-based contraction of the nuclear envelope. First, they addressed a technical issue from their previous study, finding a way to specifically impair the actomyosin contraction of the nuclear membrane without affecting the contraction of the plasma membrane. This allowed them to study the former more specifically. They then tracked individual kinetochores to reveal which were affected by nuclear membrane contraction and at what stage of displacement towards the metaphase plate. The investigation is rigorous, with all the necessary controls performed. The images are of high quality. The analyses are accurate and supported by convincing quantifications. In summary, they found that peripheral chromosomes, which are close to the nuclear membrane, are more influenced by nuclear membrane contraction than internal chromosomes. They discovered that nuclear membrane contraction primarily contributes to the initial displacement of peripheral chromosomes by moving them towards the microtubules. The microtubules then become the sole contributors to their motion towards the pole and subsequently the midplane. This step is particularly critical for the outermost chromosomes, which are located behind the spindle pole and are most likely to be missegregated.

    Significance

    While the conclusions are somewhat intuitive and could be considered incremental with regard to previous works, they are solid and improve our understanding of mitotic fidelity. The authors had already reported the overall role of nuclear membrane contraction in reducing chromosome missegregation in their previous study, as mentioned fairly and transparently in the text. However, the reason for this is now described in more detail with solid quantification. Overall, this is good-quality work which does not drastically change our understanding of chromosome congression, but contributes to improving it. Personally, I am surprised by the impact of such a small contraction (of around one micron) on the proper capture of chromosomes and wonder whether the signalling associated with the contraction has a local impact on microtubule dynamics. However, investigating this point is clearly beyond the scope of this study, which can be published as it is.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    Sheidaei and colleagues report a novel and potentially important role for an early mitotic actomyosin-based mechanism, PANEM contraction, in promoting timely congression of chromosomes located at the nuclear periphery, particularly those in polar positions. The manuscript will interest researchers studying cell division, cytoskeletal dynamics, and motor proteins. Although some data overlap with the group's prior work, the authors extend those findings by optimizing key perturbations and performing more detailed analyses of chromosome movements, which together provide a clearer mechanistic explanation. The study also builds naturally on recent ideas from other groups about how chromosome positioning influences both early and later mitotic movements.

    In its current form, however, the manuscript is not acceptable for publication. It suffers from major organizational problems, an overcrowded and confusing Results section and figures, and a lack of essential experimental controls and contextual discussion. These deficiencies make it difficult to evaluate the data and the authors' conclusions. A substantial structural revision is required to improve clarity and persuasiveness. In addition, several key control experiments and more conceptual context are needed to establish the specificity and relevance of PANEM relative to other microtubule- and actin-based mitotic mechanisms. Testing PANEM in additional cell lines or contexts would also strengthen the claim. I therefore recommend Major Revision, addressing the structural, conceptual, and experimental issues detailed below.

    Major Comments

    1. Structural overhaul and figure reorganization

    The Results section is overly dense, lacks clear structure, and includes descriptive content that belongs in the Methods. Many figure panels should be moved to Supplementary Materials. A substantial reorganization is required to transform the manuscript into a focused, "Reports"-type article. - Move methodological and descriptive details (e.g., especially from the second Results subheading and Figure 2) to the Methods or Supplementary Materials. - Remove repetitive statements that simply restate that later phenotypes arise as consequences of delayed Phase 1 (applicable to subheadings 3 onward). - Figure 4I: This panel is currently unclear and should be drastically simplified. I recommend to reorganize figures as follows: - Figure I: Keep as single figure but simplify. Figure 1D and 1E could be combined, move unnormalized SCV to supplementary materials. Same goes for 1F. - New Figure 2: Combine current Figures 2A, 3A, 3C, 3D, 4C, 4F, and 4H to illustrate how PANEM contraction facilitates initial interactions of peripheral chromosomes with spindle microtubules which increases speed of congression initiation. - New Figure 3: Combine current Figures 5A, 5C, 5D, 5F, 6B, 6C, and lower panels of 4H to show how PANEM contraction repositions polar chromosomes and reduces chromosome volume in early mitosis to enable rapid initiation of congression. - New Figure 4: Combine Figures 7A, 7B, 7D, 7E, 7F, expanded Supplementary Figure S7, and new data to demonstrate that PANEM actively pushes peripheral chromosomes inward which is important for efficient chromosome congression in diverse cellular contexts.

    1. Specificity and redundancy of actin perturbation

    To establish the specificity and relevance of PANEM, the authors should include or discuss appropriate controls:

    - Apply global actin inhibitors (e.g., cytochalasin D, latrunculin A) to disrupt the entire actin cytoskeleton. These perturbations strongly affect mitotic rounding and cytokinesis but only modestly influence early chromosome movements, as reported previously (Lancaster et al., 2013; Dewey et al., 2017; Koprivec et al., 2025). The minimal effect of global inhibition must be addressed when proposing a localized actomyosin mechanism. Comment if the apparent differences in this approach and one that the authors were using arises due to different cell types.
- Clarify why spindle-associated actin, especially near centrosomes, as reported in prior studies using human cultured cells (Kita et al., 2019; Plessner et al., 2019; Aquino-Perez et al., 2024), was not observed in this study. The Myosin-10 and actin were also observed close to centrosomes during mitosis in X.laevis mitotic spindles (Woolner et al., 2008). Possible explanations include differences in fixation, probe selection, imaging methods, or cell type. Note that some actin probes (e.g., phalloidin) poorly penetrate internal actin, and certain antibodies require harsh extraction protocols. Comment on possibility that interference with a pool of Myo10 at the centrosomes is important for effects on congression.
    1. Expansion of PANEM functional analysis

    To strengthen the conclusions and broaden the study beyond the group's previous work, PANEM function should be tested in additional contexts (some may be considered optional but important for broader impact): - Test PANEM function in at least one additional cell line that displays PANEM to rule out cell-line-specific effects. - Examine higher-ploidy or binucleated cells to determine whether multiple PANEM contractions are coordinated and if PANEM contraction contributes more in cells of higher ploidies or specific nuclear morphologies. - Investigate dependency on nuclear shape or lamina stiffness; test whether PANEM force transmission requires a rigid nuclear remnant. - Analyze PANEM's contribution under mild microtubule perturbations that are known to induce congression problems (e.g., low-dose nocodazole). - Evaluate PANEM contraction role in unsynchronized U2OS cells, where centrosome separation can occur before NEBD in a subset of cells (Koprivec et al., 2025), and in other cell types with variable spindle elongation timing. - Quantify not only the percentage of affected cells after azBB but also the number of chromosomes per cell with congression defects in the current and future experiments.

    1. Conceptual integration in Introduction and Discussion The manuscript should better situate its findings within the context of early mitotic chromosome movements:
      • Clearly state in the Introduction and elaborate in the Discussion that initiation of congression is coupled to biorientation (Vukušić & Tolić, 2025). This provides essential context for how PANEM-mediated nuclear volume reduction supports efficient congression of polar chromosomes.
      • Explain that PANEM is most critical for polar chromosomes because their peripheral positions are unfavorable for rapid biorientation (Barišić et al., 2014; Vukušić & Tolić, 2025).
      • Discuss how cell lines lacking PANEM (e.g., HeLa and others) nonetheless achieve efficient congression, and what alternative mechanisms compensate in the absence of PANEM. For example, it is well established that cells congress chromosomes after monastrol or nocodazole washout, which essentially bypasses the contribution of PANEM contraction.

    Minor Comments

    These issues are more easily addressable but will significantly improve clarity and presentation.

    Introduction

    • Remove the reference to Figure 1A in the Introduction. The portion of Figure 1 and related text that recapitulates the authors' previous work should be incorporated into the Introduction, not the Results.

    Results (by subheading)

    • First subheading: When introducing the ~8-minute early mitotic interval, cite additional studies that have characterized this period: Magidson et al., 2011 (Cell); Renda et al., 2022 (Cell Reports); Koprivec et al., 2025 (bioRxiv); Vukušić & Tolić, 2025 (Nat Commun); Barišić et al., 2013 (Nat Cell Biol).
    • Second subheading: Cite key reviews and foundational research on kinetochore architecture and sequential chromosome movement during early mitosis: Mussachio & Desai, 2017 (Biology); Itoh et al., 2018 (Sci Rep); Magidson et al., 2011 (Cell); Vukušić & Tolić, 2025 (Nat Commun); Koprivec et al., 2025 (boRxiv); Rieder & Alexander, 1990 (J Cell Biol); Skibbens et al., 1993 (J Cell Biol); Kapoor et al., 2006 (Science); Armond et al., 2015 (PLoS Comput Biol); Jaqaman et al., 2010 (J Cell Biol).
    • Third subheading: Clarify why some kinetochores on Figure 3A appear outside the white boundaries if these boundaries are intended to represent the nuclear envelope.
    • Fourth subheading: Note that congression speed is lower for centrally located kinetochores because they achieve biorientation more rapidly (Barišić et al., 2013, Nat Cell Biol; Vukušić & Tolić, 2025, Nat Commun).
    • Fifth subheading: Cite studies on polar chromosome movements: Klaasen et al., 2022 (Nature); Koprivec et al., 2025 (bioRxiv). Clarify that Figure 5F displays only those kinetochores that initiated directed congression movements.
    • Sixth subheading (currently in Discussion): Move the final paragraph of the Discussion into the Results and expand it with preliminary analyses linking PANEM contraction to congression efficiency across untreated cell types or under mild nocodazole treatment.

    Discussion

    • When discussing cortical actin, cite key reviews on its presence and function during mitosis: Kunda & Baum, 2009 (Trends Cell Biol); Pollard & O'Shaughnessy, 2019 (Annu Rev Biochem); Di Pietro et al., 2016 (EMBO Rep).

    Significance

    Advance

    This study's main strength is its novel and potentially important demonstration that contraction of PANEM, a peripheral actomyosin network that operates contracts early mitosis, contributes to the timely initiation of chromosome congression, especially for polar chromosomes. While PANEM itself was previously described by this group, this manuscript provides new mechanistic evidence, improved perturbations, and detailed chromosome tracking. To my knowledge, no prior studies have mechanistically connected this contraction to polar chromosome congression in this level of detail. The work complements dominant microtubule-centric models of chromosome congression and introduces actomyosin-based forces as a cooperating system during very early mitosis. However, the impact of the study is currently limited by major organizational issues, insufficient controls, and incomplete contextualization within existing literature. Addressing these issues will substantially improve clarity and credibility.

    Audience

    Primary audience of this study will be researchers working in cell division, mitosis, cytoskeleton dynamics, and motor proteins. The findings may interest also the wider cell biology community, particularly those studying chromosome segregation fidelity, spindle mechanics, and cytoskeletal crosstalk. If validated and clarified, the concept of PANEM could be integrated into textbooks and models of chromosome congression and could inform studies on mitotic errors and cancer cell mechanics.

    Expertise

    My expertise lies in kinetochore-microtubule interactions, spindle mechanics, chromosome congression, and mitotic signaling pathways.

  5. Version published to 10.1101/2025.09.19.677380 on bioRxiv