bims-lycede 2025-09-21 papers

Sens Actuators Rep. 2025 Jun;pii: 100259. [Epub ahead of print]9

FRET-based reporter assesses lysosomal DNA-degradation ability in live cells.

Lysosomes are multifunctional organelles that serve as the cell's central hub for metabolic signaling. Lysosomal malfunction disrupts intracellular homeostasis, leading to adverse health effects. Therefore, assessing lysosomal function is vital for advancing disease understanding and guiding therapeutic development. The existing evaluation methods rely primarily on monitoring lysosomal pH and protein degradation. Here we introduce a DNA-based reporter to evaluate lysosomal activity by assessing the DNA-degradation ability of lysosomes using fluorescence imaging. We successfully monitored the lysosomal DNA-degradation ability in dysregulated lysosomes and lysosomes in drug-induced disease model of NP-A/B and NP-C. We found that both pharmacologically induced models resulted in significant reduction in lysosomal DNA-degradation ability. This tool for monitoring lysosomal activity offers valuable insights for both therapeutic development and understanding disease progression.

Keywords: DNA-sensor; FRET; fluorescence imaging; lysosomal storage disease; lysosomes

DOI: https://doi.org/10.1016/j.snr.2024.100259

Res Sq. 2025 Sep 09. pii: rs.3.rs-7474186. [Epub ahead of print]

Processing bodies promote lysosomal quality control and cell survival during recovery from lysosomal damage.

Jingyue Cassano, Jacob Duran, Li Chen, Jing Pu, Pavel Ivanov.

Lysosomes are essential for cell survival but are highly susceptible to diverse physical and pathological stressors. Thus, the ability to initiate an acute damage response and promote recovery after stressor resolution is critical for maintaining cellular homeostasis and viability. Although recent studies have advanced our understanding of acute responses to lysosomal injury, the molecular mechanisms governing the recovery stage and distinguishing it from the acute phase remain poorly defined. Here, we delineate a key difference between these two stages in translational regulation and uncover lysosomal recovery from acute damage as a novel trigger for processing body (PB) formation. PBs are membraneless biomolecular condensates involved in RNA metabolism and translational reprogramming. We provide the first evidence that PBs are critical for lysosomal quality control and cell survival during recovery. Mechanistically, PBs are induced selectively during the recovery phase, but not during the acute damage response, through interactions with stress granules (SGs), distinct membraneless biomolecular condensates formed upon acute injury to stabilize damaged lysosomal membranes for repair. Functional analyses reveal that PBs promote lysosomal quality control by collaborating with SG-mediated membrane stabilization, while independently recruiting released cathepsins, thereby collectively supporting cell survival. Together, these findings establish PBs as central effectors of the lysosomal recovery program and underscore the broader relevance of biomolecular condensates in cellular responses to lysosomal damage and related disease processes.

DOI: https://doi.org/10.21203/rs.3.rs-7474186/v1

Nature. 2025 Sep 17.

Structural basis for mTORC1 activation on the lysosomal membrane.

Zhicheng Cui, Alessandra Esposito, Gennaro Napolitano, Andrea Ballabio, James H Hurley.

The mechanistic target of rapamycin complex 1 (mTORC1) integrates growth factor (GF) and nutrient signals to stimulate anabolic processes connected to cell growth and inhibit catabolic processes such as autophagy1,2. GF signalling through the tuberous sclerosis complex regulates the lysosomally localized small GTPase RAS homologue enriched in brain (RHEB)3. Direct binding of RHEB-GTP to the mTOR kinase subunit of mTORC1 allosterically activates the kinase by inducing a large-scale conformational change4. Here we reconstituted mTORC1 activation on membranes by RHEB, RAGs and Ragulator. Cryo-electron microscopy showed that RAPTOR and mTOR interact directly with the membrane. Full engagement of the membrane anchors is required for optimal alignment of the catalytic residues in the mTOR kinase active site. Converging signals from GFs and nutrients drive mTORC1 recruitment to and activation on lysosomal membrane in a four-step process, consisting of (1) RAG-Ragulator-driven recruitment to within ~100 Å of the lysosomal membrane; (2) RHEB-driven recruitment to within ~40 Å; (3) RAPTOR-membrane engagement and intermediate enzyme activation; and (4) mTOR-membrane engagement and full enzyme activation. RHEB and membrane engagement combined leads to full catalytic activation and structurally explains GF and nutrient signal integration at the lysosome.

DOI: https://doi.org/10.1038/s41586-025-09545-3

Adv Sci (Weinh). 2025 Sep 17. e13241

Gal3-CaN-Smurf1 Complex Sequestrates FLCN-FNIPs to Facilitate TFEB Activation in Response to Endomembrane Damage.

Qin Xia, Ziwan Liu, Gaoqing Feng, Wanting Xu, HuaHua Wang, Haihang Su, Jiaqian Li, Dan Liu, Jun Qu, Tonghui Yu, Lei Dong.

Smurf1 mediates lysosomal biogenesis upon endomembrane damage by interacting with lysosomal injury sensor Gal3 and phosphatase CaN to form Gal3-CaN-Smurf1 complex, which is critical for TFEB dephosphorylation. However, whether Smurf1 plays a role in the inhibition of mTOR-mediated TFEB phosphorylation is still unclear. TFEB phosphorylation by mTORC1 is strictly dependent on RagC/D GTPase activating protein FLCN. Here, we found that Smurf1 promotes the dissociation of RagC from TFEB upon lysosomal damage, selectively impairing TFEB phosphorylation. These findings suggest that the lysosomal damage-induced Gal3-CaN-Smurf1 complex sequesters FLCN-FNIPs to facilitate TFEB activation. This disruption of FLCN GAP function toward RagC/D impairs TFEB's lysosomal localization and phosphorylation. Notably, FLCNK462R and/or FNIP2K466R mutations reduce their binding affinity with the Gal3-CaN-Smurf1 complex, suggesting Smurf1-mediated poly-ubiquitylation of FLCNK462 and FNIP2K466 plays a role for pentamer formation. Indeed, sequestration of FLCN-FNIPs stabilizes the Gal3-CaN-Smurf1 complex, wherein Smurf1 directly binds and ubiquitinates TFEB. This facilitates TFEB's dephosphorylation and activation. These findings indicate that Gal3-CaN-Smurf1 complex interconnects with the FLCN-FNIPs to orchestrate TFEB localization and activity in response to lysosomal damage stress. Understanding Smurf1's regulation in the mTOR-TFEB axis, which balances tumor growth and stress-induced cell homeostasis, may provide novel therapeutic targets for tumor progression and drug resistance.

Keywords: FLCN‐FNIPs; Gal3‐CaN‐Smurf1 complex; TFEB; endomembrane damage; lysosomal stress; ubiquitylation

DOI: https://doi.org/10.1002/advs.202413241

bioRxiv. 2025 Sep 09. pii: 2025.09.09.674968. [Epub ahead of print]

Lysosomal RNA profiling reveals targeting of specific types of RNAs for degradation.

G Jordan Ray, Eleonora Nardini, Heather R Keys, Daniel H Lin, David M Sabatini, David P Bartel.

Autophagy targets a wide variety of substrates for degradation within lysosomes 1 . While lysosomes are known to possess RNase activity 2 , the role of lysosomal RNA degradation in post-transcriptional gene regulation is not well understood. Here, we define RNASET2, PLD3, and both endogenous and exogenous RNase A family members as lysosomal RNases. Cells lacking these RNases accumulated large amounts of lysosomal RNA. Although all types of RNA can be found within lysosomes, SRP RNAs, Y RNAs, 5' TOP mRNAs, long-lived mRNAs, and mRNAs encoding membrane and secreted proteins were specifically enriched. All types of RNA depend on autophagy for lysosomal targeting, but the lysosomally-enriched RNAs are more sensitive to loss of autophagy, implying that selective mechanisms mediate their lysosomal entry. RNA stability measurements revealed that lysosomally-degraded transcripts also had autophagy-dependent changes in stability. In exploring how specific RNAs are targeted for lysosomal degradation, we found that the Alu domain of SRP RNAs is sufficient for targeting these RNAs to lysosomes in fashion that depends on its interactions with the SRP9 and SRP14 proteins. For mRNAs, 5' TOP motifs are sufficient to increase their targeting to lysosomes for degradation in a LARP1-dependent manner. Altogether, our results establish lysosomes as selective modulators of cellular RNA content.

DOI: https://doi.org/10.1101/2025.09.09.674968