Autophagy receptor knockout effects on stress granule accumulation

PRIMARY OUTCOME

stress granule accumulation upon receptor knockout

EXPECTED OUTCOMES

Quantitative predictions: (1) WT cells: Arsenite induces 15-20 SG per cell, which clear by t1/2=3-4h after washout. By 8h, <10% of cells retain SG. (2) SQSTM1-/-: SG count 20-25 per cell (slightly more form), t1/2=6-7h (60% slower clearance). At 8h, 40-50% cells still have SG. (3) CALCOCO2-/-: Similar to SQSTM1-/-, t1/2=5-6h, 30-40% cells with SG at 8h. (4) Pathological SG (TDP-43 M337V): WT cells clear ~50% of SG by 24h. KO lines clear <20% (severe accumulation). (5) Re-expression of cognate receptor: Rescues clearance to near-WT levels (t1/2 within 1h of WT, p>0.1). (6) Bafilomycin treatment: Phenocopies KO in WT cells (t1/2 increases to 6-8h). KO + bafilomycin not additive (same pathway). (7) LC3-II accumulation during SG phase: 2-3 fold in WT, but only 1.3-1.5 fold in KO (reduced autophagy of SG).

SUCCESS CRITERIA

Primary: SQSTM1-/- or CALCOCO2-/- increases SG persistence >1.5-fold vs. WT (t1/2, log-rank test p<0.01). At 8h post-washout, >30% cells retain SG in KO vs. <10% in WT (p<0.01, chi-square). Secondary: (1) Pathological SG (disease mutants) accumulate more severely in KO lines (>2-fold retention vs. WT, p<0.01). (2) Receptor re-expression rescues phenotype (clearance kinetics not significantly different from WT, p>0.05). (3) Bafilomycin phenocopies KO (p>0.1 for WT+bafilomycin vs. KO). (4) LC3-II accumulation on SG fractions reduced >40% in KO (p<0.01). (5) Off-target validation: Top 5 predicted sites show WT sequence (no indels). (6) Consistency across 2 independent clones per genotype. Conclusion: SQSTM1 and CALCOCO2 are non-redundant receptors for SG autophagy (granulophagy).

PROTOCOL

Cell lines: Generate SQSTM1-/- and CALCOCO2-/- knockouts in U2OS cells via CRISPR-Cas9. Use dual gRNA strategy (2 gRNAs per gene) to delete coding exons. Clone and validate single-cell clones by: (1) Western blot (no protein detectable). (2) Sanger sequencing (frameshift or deletion). (3) Off-target analysis (sequence top 5 predicted off-targets). Maintain WT parental line as control. Stress granule assays: (1) Physiological SG: 0.5 mM sodium arsenite (1h), or 200 μM puromycin (1h, translation inhibitor). (2) Pathological SG: Transfect with ALS-linked mutants (TDP-43 M337V, FUS R521C) or OPMD mutant (PABPN1 A17). (3) Recovery: Induce SG with arsenite, wash out, follow for 0-8h. Quantification: Immunostain for G3BP1 (SG marker), count SG per cell (n=100 cells per genotype per condition). Measure SG size, intensity. Time-lapse imaging: Live-cell microscopy with mCherry-G3BP1, image every 30 min for 12h post-stress. Calculate SG formation rate, dissolution rate, persistence time. Rescue: Re-express GFP-SQSTM1 or GFP-CALCOCO2 in respective KO lines (lentiviral transduction). Verify rescue of SG clearance phenotype. Autophagy flux: Bafilomycin A1 (100 nM, 4h) to confirm autophagy-dependence. Western blot for LC3-II accumulation in KO vs. WT. n=3 independent experiments.

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[Autophagy receptor knockout effects on stress granule accumulation](http://scidex.ai/artifact/exp-b2f00909-1399-435d-b702-315c9ca560ee)

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