Proteomic profiling of nuclear GFP-LC3 immunoprecipitates from primary human trabecular meshwork (TM) cells identified a reproducible nuclear LC3 interactome enriched for proteins containing LC3-interacting region (LIR) and expanded LIR (xLIR) motifs. Among identified partners, clathrin heavy chain (CLTC) emerged as a previously unrecognized nuclear LC3-associated protein. CLTC localizes to the nucleus and colocalizes with nuclear LC3 puncta. Functional depletion of CLTC reduced basal LC3-II levels and markedly impaired nuclear LC3 accumulation induced by both nuclear export blockade and cyclic mechanical stretch, without altering total CLTC abundance. TM cells from glaucoma patients displayed selective impairment of mechanically induced nuclear LC3 trafficking despite comparable CLTC levels. The authors conclude that nuclear LC3 trafficking is an actively regulated, CLTC-dependent process linked to cytoskeletal and vesicular machinery and that dysregulation of this pathway may be relevant to glaucoma pathogenesis.
The authors performed mass spectrometry-based proteomic profiling of nuclear GFP-LC3 immunoprecipitates prepared from primary human TM cells. Identified proteins were analyzed for the presence of canonical LIR and expanded LIR (xLIR) motifs and for reported nuclear localization. Follow-up experiments included imaging to assess colocalization, CLTC depletion to test functional consequences, biochemical assays of LC3-II abundance, pharmacologic blockade of nuclear export with leptomycin B, and application of cyclic mechanical stretch to model mechanotransduction. TM cells derived from glaucoma patients were examined in parallel to compare responses.
Mass spectrometry of nuclear GFP-LC3 IPs yielded a reproducible set of proteins enriched for those containing LIR/xLIR motifs and reported to localize to the nucleus. The dataset defines a nuclear LC3 interactome that links LC3 to nuclear proteins and suggests potential mechanisms by which autophagy-related machinery may interact with nuclear processes. Specific protein identities beyond CLTC were reported in the profiling, but the abstract highlights motif enrichment and nuclear localization as features of the interactome.
Among the interactome members, clathrin heavy chain (CLTC) was identified as a novel nuclear LC3-associated protein. CLTC was found to localize to the nucleus in TM cells and to colocalize with nuclear LC3 puncta in imaging experiments. The data support a direct or tightly associated relationship between nuclear LC3 and CLTC in these ocular mechanosensitive cells.
Depletion of CLTC resulted in reduced basal LC3-II levels, a biochemical marker commonly associated with autophagosome abundance or formation. This finding is consistent with a role for CLTC in autophagosome biogenesis or in pathways that influence LC3 lipidation. Importantly, CLTC depletion also markedly impaired LC3 accumulation in the nucleus under conditions that normally promote nuclear LC3 accumulation, indicating a functional contribution of CLTC to LC3 nuclear trafficking.
Two experimental maneuvers that induce nuclear LC3 accumulation were studied: blockade of nuclear export using leptomycin B and application of cyclic mechanical stretch to TM cells. In both contexts, CLTC depletion significantly reduced the induced nuclear accumulation of LC3. Because total CLTC abundance was not altered by these interventions, the data imply that CLTC actively promotes LC3 nuclear translocation rather than serving only as a static scaffold.
TM cells derived from glaucoma patients showed a selective defect: mechanically induced nuclear LC3 trafficking was impaired, whereas the basal component of nuclear LC3 trafficking was not affected. Total CLTC levels in glaucomatous TM cells were comparable to control TM cells, suggesting that the defect lies in coupling the CLTC-dependent trafficking pathway to mechanotransduction or stress-responsive signaling rather than in the core transport machinery itself.
The findings position nuclear LC3 trafficking as an actively regulated process that interfaces with vesicular and cytoskeletal machinery via CLTC. By linking nuclear LC3 dynamics to mechanical stress responses and showing altered mechanosensitive trafficking in glaucoma-derived cells, the work provides a framework to explore how autophagy-related proteins influence nuclear homeostasis and how their dysregulation could contribute to disease-relevant responses in mechanosensitive ocular cells.
The study lists funding from the National Eye Institute, the BrightFocus Foundation, and Research to Prevent Blindness. The authors declared no competing interests. This report is a preprint posted on bioRxiv and has not been certified by peer review; details beyond those included in the source abstract and article materials are not reported here.