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The review is the result of a collaborative live review discussion held on 21 October 2025, organized and hosted by the PhD School in Biomedical Sciences of the University of Padova as part of the Journal Club activity. The discussion was followed by additional asynchronous work by students and the JC committee, who jointly contributed to the composition and refinement of this report.

Introduction:

This study investigates the interplay between the mTORC1 signaling and the post-translational modifications (PTM) of microtubules (MT) and how, in turn, the tyrosination or de-tyrosination of tubulin regulates lysosome dynamics and function in response to nutrients. Lysosomes shift between perinuclear and peripheral regions when nutrients are scarce or abundant, respectively, in a movement based on microtubule tracks and motor proteins. While prior work described motor recruitment to lysosomes, the authors of this paper elucidate how specific microtubule modifications influence this process. In detail, they focused on how tyrosination and de-tyrosination of microtubules differentially control lysosomal transport and mTORC1 activity, linking cytoskeletal remodeling to nutrient sensing and cancer-related signaling. Moreover, the link between mTORC1 and microtubule tyrosination or de-tyrosination may have therapeutic implications for various cancers, since abnormal mTORC1 activity, often associated with increased cell migration and epithelial to mesenchymal transition, is frequently observed in these pathological conditions.

Results:

The authors explored how the post-translational modifications of microtubules, tyrosination and de-tyrosination, regulate lysosome dynamics and mTORC1 signaling in response to nutrient availability. Using epithelial and cancer cell lines, they showed that nutrient stimulation drives lysosomes carrying active mTORC1 to move along tyrosinated microtubules toward the cell periphery, enhancing anabolic signaling. In contrast, under starvation, de-tyrosinated microtubules dominate and support autophagy. Mechanistically, active mTORC1 supposedly promotes this shift by acting through its downstream kinase S6K1, which suppresses VASH2, the enzyme responsible for the de-tyrosination of microtubules. This inhibition preserves a pool of tyrosinated microtubules and reinforces mTORC1 activity. Genetic manipulation of the enzymes TTL (responsible for tyrosination) and VASH2 confirmed that altering the PTMs balance directly affects lysosome positioning and mTORC1 activity.  In cancer models, excessive microtubule tyrosination was found to maintain mTORC1 hyperactivation and promote cell migration, while restoring de-tyrosination reduced these oncogenic traits. The authors further checked expression data from publicly available datasets and showed that, in some cancer cell lines, increased tyrosination correlates with elevated mTORC1 signaling, and that VASH2 overexpression or TTL depletion partially normalizes mTORC1 activity and reduces migratory behavior.

Methods:

To visualize and quantify the movement of lysosomes and microtubule PTMs under different conditions, the authors mainly combined super-resolution microscopy with live-cell imaging and biochemical assays. In detail, they performed the following key steps:

● Starvation and restimulation of epithelial cells (BS-C-1, derived from monkey’s kidney) to modulate mTORC1 activity;

● Quantification of the levels of tyrosinated and de-tyrosinated microtubules under starvation and restimulation;

● Tracking of lysosome motility and positioning;

● Manipulation of the enzymes responsible for tyrosination (TTL) and de-tyrosination (VASH2) to assess causal effects;

● Use of inhibitors of the mTORC1 pathway (rapamycin, PF-4708671) and mutant constructs (S6K1 variants) to dissect the signaling cascade;

● Extension of the analysis to cancer cell lines to test functional consequences for cell migration and mTORC1 hyperactivation.

Comments on each section:

The Title does convey in one sentence the key information about what was discovered, reflecting the real content of the manuscript. We note a minor typo: “mTORC1 signaling modulate” should be corrected to “mTORC1 signaling modulates…”.

The Abstract contains information about the research background, clearly states the research question as well as the approach and the key findings.

The Introduction offers a thorough presentation of the biological context: the authors clearly explain that the motors and lysosomal adaptors are known, however, if and how tubulin PTMs influence lysosomal transport and mTORC1 signaling, has not been described in detail so far. The introduction is rich in appropriate references and grounded in existing literature. Referencing seems comprehensive and balanced, not biased toward any author or laboratory. On the other hand, the main research question (“Does mTORC1 regulate microtubule tyrosination to control lysosomal transport?”) is never explicitly stated.

Materials and Methods and Results: the authors use a robust combination of super-resolution and live-cell TIRF microscopy paired with biochemical, genetic, and pharmacological approaches in their experimental strategy. However, microscopy-based quantification is limited here, since the authors’ approach is semi-quantitative. Manual scoring can be influenced by the operator, adding significant variability between experiments, and the absence of specific instructions on the procedures, such as imaging parameters, makes almost impossible their replication. Most of the experiments take advantage of the BS-C-1 cell model; however, we believe that the use of additional cell lines would have strengthened the results. For example, confirming the data in primary or mammalian epithelial cells would have made the findings more generalizable. The authors partly address this issue by including U2OS and different cancer cell lines in some experiments, but a clear justification of the specific cell line chosen and a more systematic cross-validation across the cell models would strengthen the main conclusions. Furthermore, controls are not properly described: lack of description of negative controls, positive controls or knockdown/KO controls. Other pieces of information that are missing are: incubation time, buffer composition, dilutions, etc. Finally, we think that statistical analysis needs to be improved. The authors mention quantification of lysosome motility, track displacement and fluorescence intensity with mean ± SD, but details on sample size (n) per experiment and the number of experimental replicates (biological or technical) are missing.

Discussion and Conclusions: In general, the discussion and conclusions properly convey the content of the paper. However, potential co-causalities may exist, and the authors should highlight the need of further exploring and analyzing the mechanisms under study. For instance, the observed decrease in detyrosinated microtubules upon mTORC1 activation could reflect changes in microtubule stability and lifetime rather than a direct effect on detyrosinating enzyme activity, and this possibility should be discussed.

Moreover, we have a specific concern: the authors claim that “vasohibin is directly inhibited by S6K1”, however, this is not properly correct. Indeed, this activity is inferred from PhosphositePlus, however, no experimental data supports this hypothesis. So, this inhibition should be stated more clearly as a working model, not as a demonstrated mechanism. Similarly, sentences such as “mTORC1 recruitment is sufficient to trigger lysosomal motility” overstate what is directly demonstrated, since this study does not provide a direct block of mTORC1 lysosomal recruitment nor an acute inhibition of mTORC1 kinase activity during live motility assays. We suggest rephrasing these conclusions, while clearly outlining which future experiments would be recommended.

Major issues:

● Incomplete methodological details: many protocols (fixation, staining, imaging, tracking) lack essential information (such as reagent concentrations). Suggestion: fully specify all reagents, concentrations, incubation times, temperatures, and link protocols to specific experiments.

● Lack of quantitative rigor: analyses like lysosome motility, colocalization, and TFEB nuclear localization rely on manual scoring without objective and automated measures or statistical validation. Suggestion: implement automated image analysis, define metrics clearly, and report replicates and statistical tests.

● Use of Lysotracker as lysosomal marker. A major conceptual issue is the use of Lysotracker as the primary marker to identify and track lysosomes across different nutrient conditions. Lysotracker accumulation is strongly pH-dependent, and starvation is known to increase lysosomal acidity. This means that the population of lysosomes visualized by Lisotracker may differ between conditions, potentially leading to a bias for more acidic subpopulation during starvation and confounding comparisons of motility, positioning and clustering. We encourage the authors to acknowledge this limitation and, if possible, validate key findings using pH-insensitive markers.

● Incomplete microscopy parameter information: imaging parameters, acquisition settings, and processing workflows are poorly described. Suggestion: provide detailed metadata (objectives, pixel size, z-step, exposure, laser power) and describe all the preprocessing steps.

● Insufficient controls: no clear negative/positive controls for genetic manipulation or pharmacological treatments. Suggestion: to include antibody controls for knockdown/overexpression validation or pharmacological treatments used.

● The current data do not test whether blocking mTORC1 recruitment to lysosomes or inhibiting its kinase activity is necessary for increased motility or reduced de-tyrosination. We suggest tempering statements that mTORC1 recruitment “drives” motility or “suppresses” detyrosination and instead framing these as associations that are consistent with a model in which mTORC1 contributes to these processes. The authors could also test if genetic (e.g. RAPTOR depletion) or pharmacological (e.g. Torin1, acute rapamycin) experiments would help to prove their main conclusion.

Minor issues:

● Formatting and clarity problems: Acronyms are not explicit, even at first usage (S6K1, TSC1/2 proteins, Arl8b, SKYP, JIP4, RILP48, LRRK2). Please spell them out.

● Criteria for parameter selection s: thresholds for lysosome mobility, vector cutoffs for transport direction, and selection of frames for analysis are arbitrary. Suggestion: provide rationale for all thresholds.

● Manual annotation bias: several tracking and scoring steps rely on manual observation, which is subjective.

Suggestion: use automated tracking software wherever possible.

● Limited generalizability: most experiments are conducted in a single epithelial cell line, providing no cross-validation in other cell types is recommended. Suggestion: include additional cell types or clearly describe the limitations of the study.

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.