PREreview of Decoding EGFR ligand bias through an endocytic organelle platform

This manuscript investigates the cellular and molecular mechanisms driving cell migration, specifically examining spatial-temporal dynamics across a standard 24-hour assay window. By focusing on EGFR-mediated signaling downstream of key regulatory nodes, including PLCγ2, IP3R, and RTN3, the authors attempt to delineate how distinct signaling pathways govern motility. The study provides valuable insights into directional cell movement. However, the current experimental framework and the manuscript could benefit from the suggestions below to strengthen the manuscript.

Major issues

• Missing methods section

The manuscript lacks a dedicated Methods section. Complete, detailed experimental protocols for all procedures, cell lines, reagents, and imaging pipelines must be provided to enable reproducibility.

• Unvalidated mechanistic claims (PLCγ2, IP3R, RTN3, and RAC-binding)

The manuscript highlights the roles of PLCγ2, IP3R, RTN3, and specific PLCγ2 RAC-binding domains in mediating EGFR-driven cell migration. However, direct loss-of-function or mutational experiments testing these specific components within functional migration assays are absent. The authors could either provide experimental validation (via siRNA/CRISPR knockdown, expression of RAC-binding mutants, or constitutively active RAC1 rescue experiments) during migration or refactor the text to explicitly frame these signaling nodes as logical hypotheses and future directions in the Discussion.

• Biochemical validation of the proposed signaling hierarchy

The proposed RAC1-dependent activation of PLCγ2 is well-said; however, direct evidence of biochemical interactions between endogenous RAC1 and PLCγ2 during EGF stimulation was not provided. The authors can perform co-immunoprecipitation (Co-IP) or proximity ligation assays (PLA) under EGF-stimulated conditions to confirm direct endogenous interactions.

• Distinguishing migration from adhesion dynamics

Changes in initial cell attachment, focal adhesion assembly/disassembly, and spreading rates can significantly alter cell coverage independently of intrinsic cell motility. The authors do not measure or control for baseline cell-matrix adhesion, making it unclear whether observed deficits stem from impaired cytoskeletal motility or altered substrate attachment dynamics.

• Model generalizability and functional validation

The mechanistic experiments rely primarily on HeLa cells, with limited validation in HaCaT cells or organoid models. Expanding key experiments to clinically relevant epithelial or cancer models would significantly enhance the translational impact of the findings.

The conclusions linking EGF to migration and TGFα to enhanced proliferation would be strengthened by incorporating complementary functional assays, such as scratch/wound healing, invasion, or long-term proliferation assays.

• Replication and data transparency

Certain electron microscopy (EM) and super-resolution (SIM) imaging datasets have limited independent biological replicates (n=1 in some cases). The authors must increase the number of biological replicates for these analyses and consider depositing raw microscopy images, quantitative datasets, and image-analysis scripts in a public repository to meet standard reproducibility criteria.

Minor issues

• Methodological transparency in assay media & ligand selection

The exact media composition used during the 24-hour migration assays, specifically serum concentration, must be explicitly reported, as serum directly dictates baseline proliferation and survival signals.

The authors can provide a clear rationale early in the Results section for selecting a high EGF concentration (100 ng/mL) to contextualize their choices relative to physiological ligand levels.

• Quantification and image analysis standards

The image analysis pipeline lacks explicit detail regarding thresholding parameters, ROI definitions, and whether tracking was automated or manual. Clear criteria for how gap area or individual cell trajectories were measured must be documented in the Methods section.

• Discussion of off-target interactions and streamlining

Potential crosstalk with secondary cell-surface receptors or compensatory pathways in response to high-dose stimulation/inhibition should be addressed within the 24-hour time course.

Some parts of the Discussion redundantly restate Results. The Discussion should be shortened and focused on biological interpretations, dependencies on cultured models, and study limitations (e.g., the lack of direct structural evidence).

• Terminology and definitions

Authors should define all key abbreviations upon first use in the text (e.g., NCE, CME, PM-ER contact sites, TIs, CTxB) to aid readers outside the specialized endocytosis field.

• Figure legends and western blots

Authors should simplify overly lengthy figure legends by moving detailed methodological text into the Methods section.

Also, they can ensure representative Western blots include clear molecular weight markers and present quantitative replicate data alongside representative images.

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

The authors declare that they used generative AI to come up with new ideas for their review.