Autophagy receptor identification for stress granule elimination

PRIMARY OUTCOME

identification of SQSTM1 and CALCOCO2 as stress granule autophagy receptors

EXPECTED OUTCOMES

Quantitative predictions: (1) Screen: SQSTM1 and CALCOCO2 show >70% colocalization with G3BP1 at SG periphery during recovery phase (2-4h). Other receptors show <30% colocalization. (2) SG clearance t1/2: WT cells 3-4h, SQSTM1-/- 6-8h, CALCOCO2-/- 5-7h, double KO 10-12h. (3) Receptor recruitment: SQSTM1 arrives at 1-2h post-stress, CALCOCO2 at 2-3h (sequential). (4) Co-IP: SQSTM1 pulls down G3BP1 with 5-10 fold enrichment over IgG; CALCOCO2 4-8 fold. (5) PLA: 10-20 puncta per cell at 3h recovery in WT, <2 in receptor KO. (6) Bafilomycin or ATG7 KD increases SG persistence to match receptor KO (additive effect minimal, same pathway). (7) LC3-II on SG fractions increases 3-5 fold during clearance phase in WT, but not in SQSTM1/CALCOCO2 double KO.

SUCCESS CRITERIA

Primary: SQSTM1 and CALCOCO2 show >60% colocalization with G3BP1 at 3h recovery (p<0.001, paired t-test vs. other receptors). Knockout of SQSTM1 or CALCOCO2 increases SG t1/2 by >1.5-fold (p<0.01 vs. WT, log-rank test). Secondary: (1) Co-IP enrichment >4-fold for SQSTM1 and CALCOCO2 (p<0.01). (2) PLA signal >5-fold above isotype control (p<0.001). (3) Autophagy inhibition (bafilomycin or ATG7 KD) phenocopies receptor KO (no significant difference in SG persistence, p>0.1). (4) Double KO shows additive effect (t1/2 >2x single KO, p<0.05). (5) LC3-II enrichment on SG fractions is receptor-dependent (reduced >60% in double KO, p<0.01). Reproducibility: Clearance kinetics within 30 min across 3 experiments.

PROTOCOL

Screen design: Test 8 canonical autophagy receptors: SQSTM1/p62, CALCOCO2/NDP52, NBR1, OPTN, TAX1BP1, TOLLIP, BNIP3, FUNDC1. Cells: U2OS cells expressing mCherry-G3BP1 + GFP-tagged receptors (lentiviral). Stress: 0.5 mM sodium arsenite (1h), then washout to allow SG clearance (0-8h recovery). Imaging: Live-cell confocal microscopy, image every 30 min during recovery. Quantify: (1) Receptor colocalization with G3BP1 (Mander overlap coefficient). (2) SG clearance kinetics (t1/2, plateau). (3) Receptor recruitment timing. Knockout validation: Generate SQSTM1-/-, CALCOCO2-/- using CRISPR-Cas9 (dual gRNA strategy). Verify knockout by Western blot and Sanger sequencing. Measure SG persistence in KO cells vs. WT. Interaction assays: (1) Co-IP: Pull down GFP-receptors, detect mCherry-G3BP1. (2) Proximity ligation assay (Duolink) for endogenous SQSTM1-G3BP1 and CALCOCO2-G3BP1 interactions. (3) GST pulldown with recombinant proteins to map binding domains. Autophagy dependence: Treat with bafilomycin A1 (100 nM) or ATG7 knockdown to block autophagy. Measure SG clearance in presence/absence of autophagy. LC3 lipidation: Western blot for LC3-II accumulation on SG-enriched fractions (differential centrifugation). n=4 independent experiments, 100 cells per condition.

LINKED HYPOTHESES

Prediction Markets (1 direct, 0 via hypothesis — 1 total)

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[Autophagy receptor identification for stress granule elimination](http://scidex.ai/artifact/exp-f4dcbaa9-bddd-4c2c-80c3-a7d1af45e915)

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http://scidex.ai/artifact/exp-f4dcbaa9-bddd-4c2c-80c3-a7d1af45e915

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