Journal: Advanced Science
Article Title: TBK1 Induces the Formation of Optineurin Filaments That Condensate with Polyubiquitin and LC3 for Cargo Sequestration
doi: 10.1002/advs.202509927
Figure Lengend Snippet: The M1‐ubiquitin‐specific deubiquitinase OTULIN prevents and reverses phase separation of phosphorylated Optineurin in vitro and in cells. A) Deubiquitylation activity of recombinant OTULIN. 4xM1‐ub (20 µM) was incubated with and without OTULIN (2 µM, 1 h at 37 °C) and analyzed by immunoblotting using an anti‐ubiquitin antibody (P4D1). B) OTULIN prevents the formation of pOPTN condensates. pOPTN (5 µM) and M1‐ub x chains (40 µM) were incubated with or without OTULIN (5 µM) for 60 min followed by fluorescence microscopy (3D reconstruction). Scale bar: 5 µm. C) OTULIN reverses the formation of pOPTN condensates. pOPTN and M1‐ub x were incubated for 10 or 60 min. Then OTULIN was added followed by fluorescence microscopy (3D reconstruction) after 60 min incubation in the presence of OTULIN. Scale bar: 5 µm. D) Quantification of the number of pOPTN condensates corresponding to the conditions shown in C. Data represent the mean ± SD of four independent experiments. The statistical analysis was performed using a Mann‐Whitney test (*** p ≤ 0.001). E) OTULIN suppresses the formation of Optineurin condensates in SH‐SY5Y cells. OPTN‐KO SH‐SY5Y cells generated by CRISPR/Cas9 were transiently transfected with Optineurin fused with eGFP (OPTN‐eGFP) and HA‐OTULIN or a control plasmid (‐ HA‐OTULIN) and analyzed by immunocytochemistry and fluorescence microscopy 24 h after transfection using an anti‐HA antibody. The lowest panel represents a zoom of the area marked by a square. Scale bar: 10 µm, zoom‐in scale bar: 5 µm. F) Percentage of transfected cells with pOPTN condensates corresponding to the conditions shown in E. The percentage of transfected cells (expressing OPTN‐eGFP and HA) showing at least one assembly with a diameter ≥ 0.5 µm was quantified. Three biological replicates with two fields of view each were analyzed. The bars indicate the mean ± SD and the data points of each replicate are presented as points. The statistical analysis was performed using a Mann‐Whitney test (*** p ≤ 0.001). G) Immunoblot analysis of wildtype (WT) and OPTN‐KO SH‐SY5Y cells, confirming the defective expression of Optineurin in OPTN‐KO SH‐SY5Y cells. An anti‐Optineurin antibody (HPA003279, Merck) was used for detection. H) Optineurin condensates formed in SH‐SY5Y cells co‐localize with M1‐linked ubiquitin chains. OPTN‐KO SH‐SY5Y cells transiently transfected with OPTN‐eGFP were analyzed by immunocytochemistry and fluorescence microscopy 24 h after transfection using the 1E3 anti‐M1‐ubiquitin and LC3 antibody. Scale bar: 10 µm. I) Inhibition of ubiquitylation or HOIP interferes with the formation of Optineurin condensates in SH‐SY5Y cells. OPTN‐KO SH‐SY5Y cells transiently transfected with OPTN‐eGFP were treated with the E1 ubiquitin‐activating enzyme inhibitor TAK243 (1 µM, 24 h) or the HOIP inhibitor HOIPIN‐8 (30 µM, 24 h) 4 h after transfection and then analyzed by fluorescence microscopy. Scale bar: 10 µm. J) Percentage of transfected cells with Optineurin condensates corresponding to the conditions shown in I. Cells expressing OPTN‐eGFP were classified as positive when they showed at least one assembly ≥ 0.5 µm in diameter. Five areas of view each from three biological samples were analyzed. The statistical analysis was performed using a Mann‐Whitney test (** p ≤ 0.0025).
Article Snippet: The following plasmids were obtained from Addgene: pEGFP‐N1‐OPTN‐WT (Addgene plasmid number: 27052), pEGFP‐N1‐OPTN‐Q398X(Addgene plasmid number: 68849), pEGFP‐N1‐OPTN‐E478G (Addgene number: 68848), pET28b His‐mouse UBE1 (Addgene plasmid number: 32534), pET28a‐LIC UBE2V1 (Addgene Plasmid number: 25619), pOPINF‐OTULIN (Addgene plasmid number: 61464).
Techniques: Ubiquitin Proteomics, In Vitro, Activity Assay, Recombinant, Incubation, Western Blot, Fluorescence, Microscopy, MANN-WHITNEY, Generated, CRISPR, Transfection, Control, Plasmid Preparation, Immunocytochemistry, Expressing, Inhibition
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