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In this manuscript, Hari-Gupta et al. investigate the regulation of estrogen receptor (ER)-mediated signalling by nuclear myosin VI, explore the molecular basis of this regulation, and examine its implications for ER-positive breast cancer therapy. The authors combine advanced microscopy with genetic and functional approaches, generating an extensive dataset that highlights myosin VI as a potential therapeutic target in ER-positive breast cancer. The topic is timely and of broad interest.

While the manuscript presents intriguing observations, several aspects of the experimental design, data interpretation, and reporting leave some uncertainty regarding the proposed underlying mechanism. My main concern is that, despite clinical evidence showing correlated expression of ER and myosin VI, the authors do not appear to consider—or control for—the overall expression levels of these proteins in many of their key experiments. In several cases, the absence (or lack of description) of appropriate controls leaves room for alternative interpretations. Addressing the points below would greatly clarify and strengthen the conclusions.

Re: subsection “The expression levels of myosin VI and estrogen receptor are correlated across breast cancer tissues.”

1. Since MYO6 up-regulation is cancer-type specific, it would be informative to test whether the observed stage independence remains when the analysis is stratified by breast cancer subtype (Supplementary Fig. 1B). I assume all subtypes were pooled in this analysis, as this is not explicitly stated.

2. Additional remarks:

   • Fig. 1A: As the study focuses on breast cancer, this panel may distract from the central narrative. If the authors consider this panel essential, clearer annotation would help: several bar pairs lack labels, abbreviations are not defined, and statistical significance between tumour and normal samples is not shown.

   • Fig. 1A & 1B: Annotation appears inconsistent with legend (“expression level” vs “log2 expression”).

   • The ATCC HTB-20 cell line is BT-474, not BT-464.

Re: subsection “The myosin VI and estrogen receptor interaction is required for nuclear localisation.”

1. Across multiple panels, the most striking effect of several treatments appears to be variation in overall protein levels, which is not discussed, even though clinical datasets indicate a strong correlation between ER and MYO6 expression. This seems particularly relevant to Fig. 2A but also applies to later figures.

2. Fig. 2A:

   • I do not observe a clear difference in myosin VI nuclear localisation between conditions.

   • Conversely, ER appears largely nuclear in the myosin VI KD/KO conditions, despite lower overall expression, which seems inconsistent with the proposed model that ER nuclear localisation requires myosin VI.

   • The ER siRNA panels appear blurred, making direct comparison difficult.

3. Fig. 2B: The nuclear fraction of myosin VI appears higher under stimulated conditions; however, controls demonstrating successful fractionation and total protein levels are not shown.

4. Fig. 2C–E: Without a description of the image analysis pipeline, it is difficult to assess whether differences could arise from analysis-dependent artefacts.

5. Statistical considerations (Fig. 2D & 2E): Given the very small p-values, it seems possible that individual cells rather than experimental replicates were used as independent measurements, although the legend suggests n = 3 (independent experiments). Clarification would be helpful.

6. Choice of cell line: Fig. 1H suggests that ER and myosin VI localisation relationships may differ across cell lines. It is therefore not entirely clear why only MCF7 cells were selected to examine potential interdependency.

7. Additional remarks:

• The subsection title is ambiguous: it is not immediately clear which localisation depends on the ER–myosin VI interaction, nor whether a direct or indirect interaction is implied.

• A description of the image analysis workflow is missing, making interpretation of multiple figures challenging.

Re: subsection “The sub-nuclear organisation of the estrogen receptor is regulated by active myosin VI”

1. There are notable discrepancies between confocal (Fig. 2A) and STORM (Fig. 3A) images:

   • Under basal and stimulated conditions, myosin VI appears predominantly nuclear in STORM but largely cytoplasmic in confocal images.

   • In TIP and siMVI conditions, ER appears strongly nuclear in confocal images, yet nuclear enrichment is not apparent in STORM.

2. Cluster and colocalisation analysis:

• It is unclear how differences in protein concentration were controlled for; higher concentration alone could increase the apparent proximity of molecules.

• The analysis uses n = 10 ROIs but does not state the number of independent experiments, making statistical interpretation uncertain.

1. Single-particle tracking:

• Changes in diffusion may reflect differences in cluster size, macromolecular crowding, or nuclear viscosity rather than transcriptional complex formation.

• STORM data indicate formation of large clusters; could these alone explain the decreased diffusion?

• Several treatments visibly alter nuclear size (e.g., Fig. 2A and Fig. 4D). Differences in nuclear volume or cell-cycle distribution could significantly affect nucleoplasmic viscosity. It would be helpful to know whether such factors were ruled out.

• Summary diffusion constants are shown, but the distribution of track-level diffusion values could provide further insights.

Additional remark:

• The text in this subsection of the PDF appears misaligned: paragraphs switch between columns.

Re: subsection “The interdependency of myosin VI – ER functionality impacts large-scale chromatin architecture”

Fig. 4D: Some nuclei display more than four spots per gene and variable signal intensity. If gene amplification is known or suspected, clarification would be helpful, as would an explanation of how specific vs nonspecific signals were distinguished.

Re: subsection “Myosin VI regulates ER-driven gene expression”

As discussed above, several treatments seem to alter ER and myosin VI expression levels and nuclear accumulation. It is therefore unclear whether changes observed in ChIP reflect specific enrichment or simply altered nuclear abundance.

Additional remarks:

• RNA-seq methods list WT, siMVI, and siScr conditions; the reference used for differential expression analysis is not specified.

• Statistical significance for GO categories (Fig. 5B-C, Supplementary Table 1) is not indicated.

• Statistical significance for RT-qPCR data (Fig. 5D) is also not shown.

• GREB1, an ER target examined elsewhere, is absent from the RT-qPCR panel.

• Experimental conditions differ between RT-qPCR (estradiol + MVI inhibition) and ChIP (MVI inhibition only) without explanation.

Re: subsection “Myosin VI perturbation impedes cell growth and migration”

The authors attribute changes in proliferation to ER activity downstream of myosin VI, but this is not directly demonstrated; ER-independent explanations remain possible.

Additional remarks:

• “Cell growth” and “cell proliferation” are used interchangeably; consistent terminology would reduce ambiguity.

• The connection between Supplementary Fig. 7B and the functional assays is unclear, especially since combined siMVI + TIP treatment was not examined in the assays.

• A uniform ~2.5-fold decrease in expression across all genes and conditions is unusual; could this reflect a systematic issue (e.g., plate-position effects)?

• In Fig. 5D, TFF1 decreases ~10-fold, whereas VEGFA and WNT4 decrease ~13–14-fold, so this may be coincidental, but clarification would help.

Re: subsection “Targeting myosin VI in combination with endocrine therapy is more effective than monotherapy”

1. Fig. 7C vs 7D: 0.1 µM fulvestrant appears to cause substantial ER redistribution in Fig. 7C but little change in Fig. 7D; moreover, nuclear exclusion seems stronger at 0.1 µM than at 1 µM, which is not reflected in the quantification. Clarification may be needed.

2. According to Fig. 7D, 0.1 µM fulvestrant reduces nuclear MVI more strongly than nuclear ER, which differs from the proposed reciprocal behaviour.

3. Tamoxifen and fulvestrant have opposite effects on ER/MVI localisation (Fig. 7A–D) yet both suppress ER-target gene expression and cell viability (Fig. 7E–F). How this aligns with the model of nuclear myosin VI facilitating ER transcriptional activity is not fully clear.

Re: subsection “Myosin VI inhibition overcomes endocrine-resistant ER mutants”

1. Since constitutively active ERα mutants model endocrine-resistant tumours, it would be valuable to show that they indeed behave independently of ligand and antagonist in this system.

2. TAM+TIP and FUL+TIP reduce viability to ~25% of control in Fig. 7F but only to ~50% in Fig. 8C for ERα mutants. Further explanation of how this fits with the proposed role of myosin VI would strengthen the argument.

Additional remark:

• The statement that TIP reduces “target gene expression to near baseline” would benefit from defining what “baseline” refers to in this context.

Competing interests

The author declares that they have no competing interests.

Use of Artificial Intelligence (AI)

The author declares that they did not use generative AI to come up with new ideas for their review.