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. Author manuscript; available in PMC: 2023 Oct 23.
Published in final edited form as: Cell Rep. 2023 Jan 25;42(2):112037. doi: 10.1016/j.celrep.2023.112037

Figure 7. ALS-associated mutations in p62 perturb lysophagy.

Figure 7.

(A) Schematic of p62 domain architecture, highlighting specific ALS-associated mutations in p62 (L341V, P392L, and G425R).

(B) Representative images of α-LAMP1 in HeLa that were transiently transfected with p62 siRNA (#1) alongside either WT-p62, L341V, P392L, G425R, or mCherry vector after addition of 750 μM LLOMe for 2 h. Cell outlines were traced. SBs: 10 μm.

(C) Quantification of α-LAMP1 area overlapping with p62 area across different conditions (one-way ANOVA, p(WT/L341V) < 0.01, p(WT/P392L) < 0.001, p(WT/G425R) < 0.001, p(WT/mCherry) < 0.0001, n = 3 experiments. Number of cells analyzed per condition ≥ 22).

(D) Representative images of α-LC3B in HeLa cells that were transiently transfected with p62 siRNA (#1) alongside FLAG-LAMTOR2 and either WT-p62, P392L, G425R, or mCherry after addition of 750 μM LLOMe for 2 h. Merged channel shows LAMTOR2 and LC3B only. Cell outlines were traced. SBs: 10 μm.

(E) Quantification of normalized α-LC3B intensity at LAMTOR2 puncta across different conditions (Kruskal-Wallis test, p(WT/P392L) < 0.01, p(WT/G425R) < 0.05, p(WT/mCherry) < 0.0001, n ≥ 49 cells per condition.

(F) A model for p62-dependent lysophagy. p62 is recruited to damaged lysosomes through ubiquitin. Accumulation of p62 on damaged lysosomes results in phase separation. This phase separation of p62 is critically regulated by the small heat shock protein HSP27. HSP27 maintains the liquidity of p62 condensates, facilitating engulfment by autophagosomes.

All error bars reflect mean ± SEM. See also Figure S7.