مراجعة PREreview لMechanoresponsive modulation of nuclear pore complex structure and function by O -GlcNAc

Overall assessment

The manuscript “Mechanoresponsive modulation of nuclear pore complex structure and function by O-GlcNAc” by Chandra et al. studies how nuclear pore complex (NPC) permeability is adapted to different cell type and mechanical environments. The authors show passive NPC diffusion barrier varies across different cell types, especially the particularly stringent passive diffusion barrier in neuronal models, which is associated with lower nucleoporin (NUP) O-linked N-acetylglucosamine (O-GlcNAc) modification level. The correlation between GlcNAcylation and permeability is confirmed by NPC-targeted OGT/OGA tools and their inhibitors. Using pan-Expansion Microscopy, the authors further show increasing GlcNAcylation dilates the NPC. Interestingly, cells can increase NUP GlcNAcylation to counteract the NPC constriction under mechanical inputs including substrate stiffness and hyper-osmotic shock. The manuscript demonstrates to the field the different NUP GlcNAcylation across different cell types, and the strong correlation on the coupling between NUP GlcNAcylation and permeability of NPC diffusion barrier. However, the current evidence did not support the causality mechanism that GlcNAcylation alters the diffusion barrier permeability. Several conclusions could be strengthened with controls and supporting evidence.

Major comments

1. Insufficient evidence for the causality between NUP GlcNAcylation and NPC diffusion permeability. The data do not prove that NUP GlcNAcylation directly modulates the NPC diffusion barrier. To further clarify the causal mechanism, it will help by systematic mapping of GlcNAcylation on nucleoporins via mass spectrometry, followed by specific mutagenesis validation in reconstituted systems and functional analysis in cells. Alternatively, the conclusion could be phrased cautiously to reflect the correlation between NUP GlcNAcylation and passive permeability, rather than implying a causality mechanism.

Minor comments

1. The specificity of NPC-targeted OGT/OGA could be strengthened by further validation. The authors utilize RL2 signal to compare the GlcNAcylation profiles in the presence or absence of Halo-GBP-OGT. However, the RL2 antibody recognizes GlcNAc on proteins with preference for GlcNAcylated NUPs. To clarify the changes of global GlcNAcylation of whole-cell, a second complementary antibody such as CTD110.6 can characterize broader protein GlcNAcylations. The selectivity of these tools for NPC modification can be supported with this data.

2. Untransfected control could help with Halo-GBP-OGA comparisons in Figures 4E, 4F and 4H. A catalytically inactive Halo-GBP-OGT control could be included in Figures 4B and 4C. These complementary data will help readers understand the GlcNAcylation efficiency by engineered OGA/OGT tools.

3. The connection between nucleoporin O-GlcNAcylation and NPC dilation by pan-Expansion Microscopy (Figure 5) could be clarified by further validation. The OGT/OGA inhibitors can have global indirect effects on NPC. To clarify this, comparing NPC dilation following OSMI-4/TMG treatment with catalytically active and inactive Halo-GBP-OGT/OGA would provide stronger support for the proposed mechanism.

4. The response of NUP GlcNAcylation to different substrates could be substantiated if consistent stiffness conditions are used. The authors compare 3-kPa and 1.5-MPa substrates in Figure 6A, 6D, and 6E. However, plastic and 2-kPa substrates are used in Figure 6B and 6C. Using consistently matched substrate conditions across these experiments would help readers follow the functional results.

5. The active transport across NPC is not discussed. The manuscript primarily assesses the effects of O-GlcNAcylation on passive diffusion across NPCs. If additional active nuclear import/export experiments are not performed, please consider expanding the discussion of the implications for active nucleocytoplasmic transport. With this discussion, readers can have a complete view of how NUP GlcNAcylation can both modulate active and passive nucleocytoplasmic transport across NPC.

6. Reference control subunit is not consistent in Western blot (WB) analysis. NUP62 is used in Figures 3A and S1B, whereas NUP93 is used in Figures 3D, 6B, and 6C. A corresponding NUP62 or NUP93 reference can help readers to follow Figures S2A and S2D. In Figure S1B, NUP93 would provide a more appropriate reference because the experiment includes NUP62 siRNA. Providing more rationale in the methods for using NUP62 versus NUP93 as reference proteins in the WB could make the story easier to follow.

7. Different cell lines used in different parts of paper makes it difficult to assess whether the conclusion can be generalized across cell types. Providing the rationale for using different cell lines across the experiments could help improve. Specifically, briefly state why U2OS-NUP96-GFP cells were selected for targeted enzyme recruitment, iPSCs for pan-ExM, and U2OS/A549 cells for the substrate-stiffness experiments.

7. Consider presenting the broad cell-type comparisons in Figure 2E earlier, for example within Figure 1. Introducing the differences across cell types before focusing on neuronal models could improve the narrative flow.

8. The title could be more explicit. “O-GlcNAcylation modulates nuclear pore complex permeability and dilation” would more directly describe the scope of the study.

I am one of 2 participants from the same research lab that reviewed this preprint as part of the Transparent and Accountable Peer Review training program. This report was written with the support of my research mentor.

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

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