The microtubule GTP-tubulin cap size is modulated during cell division

Name: Verified Rigor Record — The microtubule GTP-tubulin cap size is modulated during cell division
Item: The microtubule GTP-tubulin cap size is modulated during cell division
Rating: 90
Author: Αlpha¹

Cassidy, A. C.; Burnette, D. T.; Zanic, M.

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Αlpha¹ Rigor Review

Rigor Review Verified

Critical

High-priority

Action items to resolve · Critical / High

Confidence: moderateEngine: 7.6.0Source: full text

Not verified in this report

No reported statistical tests were found to recompute.

This Alpha1 Rigor Review uses AI Rigor Reviewers trained on a curated corpus of high-fidelity and retracted papers, with expert supervision and curation. It can still make mistakes; verify each finding against the source before relying on it.

The paper is methodologically sound in its core scientific premise, study design, and statistical reporting, with clear replication across cell lines and detailed methods. Major weaknesses are the complete absence of data and code sharing, and missing cell line authentication and mycoplasma testing, which are important for reproducibility.

Three independent reviewers evaluated the paper across eight dimensions. The reviewers diverged on study design (pass vs warn vs not applicable) and key resources (pass vs warn), but consensus was reached after weighing evidence. The statistics verification component found no tests to recompute, so no statement about numerical correctness is made. The citation verification flagged 9 references not found in any registry, which is a potential integrity concern.

12 major claims checked against the paper's own evidence: all adequately supported.

partialConclusions, paragraph 1Reviewers 1, 2, 3
Modulation of GTP-cap size is a cell-cycle-regulated mechanism for tuning microtubule stability.
The paper shows that cap size changes across the cell cycle, but does not directly measure microtubule stability or catastrophe rates; the link to stability is inferred from prior literature. The claim is partially supported by the correlative evidence.
Evidence: The discussion cites prior work linking cap size to catastrophe, but the present study only measures cap size, not stability directly.
“our results identify modulation of the microtubule GTP-cap size as a potentially powerful mechanism to tune microtubule stability throughout the cell cycle”
partialConclusionsReviewer 2
The reduction in GTP-cap size is global rather than spatially regulated within the cell.
The claim is supported for metaphase and anaphase astral vs spindle microtubules, but the paper does not test spatial regulation in other mitotic stages (e.g., prometaphase) or other cellular contexts (e.g., within the spindle itself). The cautionary wording ('argues against') in the paper appropriately reflects this.
Evidence: Figure 3D and 3G for metaphase and anaphase.
“This result argues against spatially restricted mechanisms, such as localized force generation or compartmentalized MAP activity, as primary drivers of the GTP-cap size modulation.”
supportedResults, GTP-cap size is reduced during mitosisReviewer 1
GTP-cap size is reduced during mitosis for matched microtubule growth rates.
The paper directly compares EB1 comet lengths for matched growth rates and finds statistically significant differences between interphase and mitotic stages, as shown by linear regression with p<0.0001.
Evidence: Figure 2G and 2H show reduced EB1 comet lengths at matched growth rates in prometaphase, metaphase, and anaphase relative to interphase, with statistical tests confirming differences.
“However, when these data were fit to a linear regression, we found that this relationship—specifically the slope—is not conserved across stages (; p-value < 0.0001).”
supportedResults, GTP-cap size is not spatially regulated in metaphase or anaphaseReviewer 1
The relationship between GTP-cap size and microtubule growth rate is not spatially regulated in mitosis.
The paper compares astral and spindle microtubules in metaphase and anaphase and finds no significant difference in slopes (p=0.27, p=0.43), supporting the claim.
Evidence: Figure 3D and 3G show overlapping linear fits and p-values >0.05.
“this relationship between the EB1comet length and the microtubule growth rate is not spatially regulated in metaphase (; slope of 0.68 ± 0.11 s [95% CI] for spindle vs. 0.78 ± 0.19 s [95% CI] for astral, p-value = 0.27).”
supportedResults, Changes in GTP-cap size are conserved across cell typesReviewer 1
Changes in GTP-cap size are conserved across cell types.
The paper replicates the experiment in a second cell line (PtK1) and finds similar patterns of cap size reduction during mitosis, with most stages showing the same qualitative trends.
Evidence: Figure 4 shows similar linear relationships and reduces cap sizes in mitotic stages for PtK1 cells, with statistical tests confirming differences.
“in agreement with LLC-PK1 data, the relationship between microtubule growth rate and GTP-cap size for different cell cycle stages could not be described by a single linear regression model (; p-value < 0.0001).”
supportedConclusions, paragraph 3Reviewer 1
The reduction in GTP-cap size during mitosis is regulated globally rather than locally.
The spatial analysis shows no difference between astral and spindle microtubules in metaphase or anaphase, supporting a global regulation mechanism.
Evidence: Figure 3 and associated p-values (0.27, 0.43) show no spatial difference.
supportedResults, GTP-cap size is reduced during mitosisReviewer 2
GTP-cap size is reduced during mitosis relative to interphase for matched growth rates.
The claim is directly supported by the data shown in Figure 2 and statistical analysis showing significant differences in the linear regressions across stages (p < 0.0001) and reduced comet lengths in mitotic stages.
Evidence: Figure 2G-H and the statistical F-test results: p < 0.0001 for different slopes across stages.
“we found that this relationship—specifically the slope—is not conserved across stages (; p-value < 0.0001). Instead, for matched microtubule growth rates, EB1 comet length gradually decreased from interphase through prophase to prometaphase.”
supportedResults, GTP-cap size is not spatially regulated in metaphase or anaphaseReviewer 2
GTP-cap size is not spatially regulated in metaphase or anaphase (astral vs spindle microtubules).
The claim is supported by statistical comparisons of slopes for astral vs spindle microtubules, with p-values of 0.27 and 0.43, indicating no significant difference.
Evidence: Figure 3D and 3G, with slope comparisons: p = 0.27 for metaphase, p = 0.43 for anaphase.
“the relationship between the EB1comet length and the microtubule growth rate is not spatially regulated in metaphase (, slope of 0.68 ± 0.11 s [95% CI] for spindle vs. 0.78 ± 0.19 s [95% CI] for astral, p-value = 0.27).”
supportedResults, Changes in GTP-cap size are conserved across cell typesReviewer 2
Changes in GTP-cap size are conserved across cell types (LLC-PK1 and PtK1).
The claim is supported by parallel experiments in PtK1 cells showing the same pattern of reduced comet lengths in mitosis and similar statistical distinctions between stages.
Evidence: Figure 4 and accompanying analysis: 'EB1 comets were generally smaller during mitosis' and p < 0.0001 for different linear models across stages.
“in agreement with LLC-PK1 data, the relationship between microtubule growth rate and GTP-cap size for different cell cycle stages could not be described by a single linear regression model (; p-value < 0.0001).”
supportedAbstract; Results, 'GTP-cap size is reduced during mitosis'Reviewer 3
GTP-cap size for matching growth rates is reduced during mitosis.
The paper presents direct evidence from LLC-PK1 and PtK1 cells showing that for matched growth rates (100–320 nm/s), EB1 comet lengths (proxy for GTP-cap) are smaller in mitosis compared to interphase.
Evidence: Figure 2 panels A-F and G-H show the data; text reports p-value < 0.0001 for F-test rejecting a single linear model across stages.
“Our data reveal that GTP-cap size for matching growth rates is reduced during mitosis.”
supportedAbstract; Results, 'GTP-cap size is not spatially regulated in metaphase or anaphase'Reviewer 3
The scaling between GTP-cap size and microtubule growth rate is not spatially regulated in mitosis.
The paper tests astral vs. spindle microtubules in metaphase and anaphase, finding no significant difference in the slope (metaphase p=0.27, anaphase p=0.43), directly supporting the claim.
Evidence: Figure 3 panels D and G show overlapping slopes with 95% CIs; p-values stated in figure legends.
“Comparison of EB1 comets on astral versus spindle microtubules reveals that the scaling between the GTP-cap size and microtubule growth rate is not spatially regulated in mitosis.”
supportedAbstract; Results, 'Changes in GTP-cap size are conserved across cell types'Reviewer 3
These regulatory patterns are conserved across epithelial cells from two different species.
The paper repeats the entire analysis in PtK1 cells (potoroo) and obtains consistent results: mitosis-associated reduction in cap size and no spatial regulation pattern, supporting cross-species conservation.
Evidence: Figure 4 panels A-H show analogous data for PtK1 cells; text reports 'conserved across cell types' and an F-test p<0.0001 for stage differences, and successful grouping of similar stages.
“Taken together, these findings suggest that modulation of the GTP-cap size represents a general mechanism for mitotic-specific regulation of microtubule stability and dynamics.”

Rigor

Eight Dimensions5 pass · 2 warn

12 major claims2 partial10 supported

8 copyedit issues flagged: mostly clarity, punctuation, typo.

Checked 78 references: 69 verified — 9 unresolved.

Reproducibility

1 data/code link checked; 1 live.

datahttp://n2t.net/addgene:55055LIVEHTTP 200
Resolves, but the content could not be matched to the paper.

Alpha¹ Rigor Action ItemsFinal pass before journal submission

As a post-publication audit, the paper has several robustness gaps: the lack of data/code availability precludes independent verification of the results; the missing cell line authentication and mycoplasma testing raise concerns about reproducibility; and the 9 unfound references may indicate citation errors or fabrication. An informed reader should weigh these issues when assessing the reliability of the conclusions. A correction or independent re-analysis of the key findings would strengthen confidence.

1.
HIGHdata code
Add a data availability statement specifying a repository (e.g., BioImage Archive, Zenodo) with a DOI for the raw imaging data and processed analysis outputs, and deposit the data.
Without a data availability statement and repository deposit, the study cannot be independently verified, and this is a major transparency gap.
2.
HIGHdata code
Deposit the custom MATLAB analysis scripts in a public repository (e.g., GitHub, Zenodo) with a permanent identifier and cite them in the manuscript.
The analysis relies on custom code; sharing it is essential for computational reproducibility.
3.
HIGHrigor
Verify the 9 references that were not found in any registry (e.g., 'Molecular Biology of the Cell', 'Interference of GTP hydrolysis in the mechanism of microtubule assembly: an experimental study') and either correct or remove them; if they are genuine, provide proper citation details.
Unresolved references may be fabrication signals or citation errors, undermining the paper's credibility.
4.
HIGHreporting
Report the sex of the cell lines used (LLC-PK1 is derived from a male pig, PtK1 from a female potoroo) or state that the sex is unknown/not determined.
Sex is a biological variable that can influence cell behavior; reporting it supports reproducibility.
5.
HIGHreporting
Include a statement on cell line authentication (e.g., STR profiling) or state that the lines were obtained from a trusted source and used within a limited passage number.
Authentication ensures the cell lines are genuine and not cross-contaminated, a standard requirement for cell culture studies.
6.
HIGHreporting
Report mycoplasma testing status for the cell lines, including the method and result.
Mycoplasma contamination can alter cell behavior and is a common source of irreproducibility.
7.
MEDIUMstatistics
Verify and report the assumptions of linear regression (normality of residuals, homoscedasticity) for the models relating comet length to growth rate, or note that robust methods were used.
Without verifying assumptions, the validity of the p-values and confidence intervals is uncertain.
8.
MEDIUMstatistics
Replace the inequality p-values (e.g., 'p < 0.0001') with exact p-values (e.g., 'p = 3.2e-6') for all comparisons, especially the global F-test.
Exact p-values allow readers to assess the strength of evidence more precisely.
9.
MEDIUMreporting
Define biological vs. technical replicates explicitly in the Methods; state that the data from 3 cells per day over 3 days represent biological replicates.
Clear replicate definition is critical for understanding the statistical independence of observations.
10.
MEDIUMreporting
Add an a priori power analysis or sample-size justification for the number of cells and growth events per cell-cycle stage.
Power analysis demonstrates that the study was designed to detect meaningful effects.
11.
LOWcopyedit
Fix the missing space in 'EB1comet length' (should be 'EB1 comet length') in the Results section.
Typographical error that could cause confusion.
12.
LOWcopyedit
Replace all empty parentheses '()' (appearing in multiple places, e.g., Results paragraph 1, Methods paragraph 1) with the intended figure/table/citation references.
Empty parentheses are placeholders and indicate incomplete formatting.
13.
LOWcopyedit
Correct the sentence in the Introduction: 'disassembly of the interphase microtubule cytoskeleton is completely disassembled' to avoid redundancy, e.g., 'disassembly of the interphase microtubule cytoskeleton is complete'.
Minor word repetition that reduces readability.
14.
LOWcopyedit
Add parentheses to the CTCF formula in the Methods: 'CTCF = IntegratedDensity_cellROI − (Area_of_cellROI × MeanGrayValue_of_backgroundROI)'.
Clarifies the calculation order and matches standard formula notation.
15.
LOWreporting
Reference a relevant reporting guideline (e.g., ARRIVE for cell-based studies, or a journal-specific checklist) in the Methods section.
While not mandatory, referencing a guideline demonstrates adherence to reporting standards.

Full text (55,912 chars)Analyzed: Jul 7, 2026

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