Journal: eLife

Article Title: Degradation of engulfed mitochondria is rate-limiting in Optineurin-mediated mitophagy in neurons

doi: 10.7554/elife.50260

Figure Lengend Snippet: Figure 2. OPTN colocalizes with upstream mitophagy-associated proteins within an hour of antioxidant removal. (A) Schematic of the translocation of mitophagy-associated proteins to a damaged organelle. (B) Volume rendering of an OPTN-positive mitochondrion. Scale bar, 0.5 mm. (C) Representative image of an OPTN-positive mitochondrion that is TMRE-negative, confirming specific recruitment to damaged organelles. Scale bar, 1 mm. (D– F) Representative somal images of neurons expressing markers for mitochondria, OPTN, and ubiquitin (Ub; D), Parkin (E), and TBK1 (F). After 1 h in AO-free media, translocated proteins form rings around damaged mitochondria (1; red arrows and inset) and form puncta that colocalize with damaged mitochondria (2; green arrows and inset). Scale bar, 5 mm. The online version of this article includes the following figure supplement(s) for figure 2:

Article Snippet: DOI: https://doi.org/10.7554/eLife.50260 20 of 30 was generated from site-directed mutagenesis, untagged-Parkin (mCherry-Parkin was removed), YFP-NavII-III (Addgene 26056), GFP-Ub (Addgene 11928), TBK1 (Addgene 23851) was recloned into a pSNAPf vector (New England Biolabs), LAMP1-EGFP was subcloned from Addgene 1817 where RFP was replaced with EGFP, LAMP2-EGFP (provided by E. Chapman, University of Wisconsin-Madi- son was subcloned into pEGFP_N1), SEP-LAMP1-RFP (kindly provided by J. Bonifacino, NIH, Bethesda) and SNAP-WIPI2B (subcloned from GFP-WIPI2B).

Techniques: Translocation Assay, Expressing, Ubiquitin Proteomics

Journal: eLife

Article Title: Degradation of engulfed mitochondria is rate-limiting in Optineurin-mediated mitophagy in neurons

doi: 10.7554/elife.50260

Figure Lengend Snippet: Figure 9. Model depicting the spatial and temporal regulation of OPTN-mediated neuronal mitophagy. (A) Upon mitophagy induction, Parkin translocates to spherical mitochondria and increases the abundance of ubiquitin chains. OPTN and its kinase TBK1 are recruited followed by sequestration and elimination via autophagosome engulfment and lysosomal fusion, as monitored by LC3 and LAMP1/LysoT, respectively. This quality control mechanism is compartmentally restricted to the soma and rarely occurs in the axon. As a result, other quality control pathways may regulate axonal mitochondria. (B) Parkin, Ub, TBK1 and OPTN localize with damaged organelles within an hour of inducing mitophagy. LC3 translocation occurs after OPTN and ~75% of OPTN-positive mitochondria are LC3-positive, forming mitophagosomes an hour after initial damage. Under basal conditions, ‘late’ mitophagosomes (OPTN-LAMP-positive mitochondria) routinely form within an hour. However, mitophagy induction perturbs this pathway, increasing the number of LAMP1-negative OPTN-positive mitochondria. Interestingly, only a small fraction of OPTN-positive mitochondria are acidified in either basal or induced conditions, suggesting lysosomal acidification to eliminate damaged organelles is rate-limiting.

Article Snippet: DOI: https://doi.org/10.7554/eLife.50260 20 of 30 was generated from site-directed mutagenesis, untagged-Parkin (mCherry-Parkin was removed), YFP-NavII-III (Addgene 26056), GFP-Ub (Addgene 11928), TBK1 (Addgene 23851) was recloned into a pSNAPf vector (New England Biolabs), LAMP1-EGFP was subcloned from Addgene 1817 where RFP was replaced with EGFP, LAMP2-EGFP (provided by E. Chapman, University of Wisconsin-Madi- son was subcloned into pEGFP_N1), SEP-LAMP1-RFP (kindly provided by J. Bonifacino, NIH, Bethesda) and SNAP-WIPI2B (subcloned from GFP-WIPI2B).

Techniques: Ubiquitin Proteomics, Control, Translocation Assay

Journal: ESC heart failure

Article Title: Rnf144b alleviates the inflammatory responses and cardiac dysfunction in sepsis.

doi: 10.1002/ehf2.14383

Figure Lengend Snippet: Figure 4 Rnf144b was required for TBK1 activation and suppressed the activity of NF-κb. (A) The interacted proteins of Rnf144b were presented in STRING Interaction Network. (B) Immunoassay of HEK293T cells transfected with various combinations of expression vectors for FLAG-tagged TBK1 (FLAG-TBK1) and HA-tagged RNF144B (HA-RNF144B) followed by immunoprecipitation of proteins from lysates with anti-FLAG and immunoblot anal- ysis with HA. (C, D) The WT and RNF144b-deficient BMDMs were stimulated with LPS (1 μg/mL) for indicated time points. IB analysis of the indicated phosphorylated (P-) and total indicated proteins in whole-cell lysates. (E) After LPS stimulation, BMDMs were lysed for co-IP assay. The ubiquitination level of TBK1 protein was determined by IB. (F) The WT and RNF144b-deficient BMDMs were stimulated with LPS (1 μg/mL) and TBK1 inhibitor Amlexanox (2 μM) for indicated time points. The expression of induced cytokines of WT and RNF144b-deficient BMDMs were measured by qRT- PCR. *P < 0.05.

Article Snippet: Antibodies targeting phospho-TBK1 (Ser172, D52C2, 1:1000), GAPDH (D16H11, 1:1000), p65 (C22B4, 1:1000), histone 3 (D1H2, 1:1000), and ubiquitin (P4D1, 1:1000) were purchased from Cell Signaling Technology Inc. Antibodies targeting RNF144b (PA5-39152, 1:1000) were purchased from Invitrogen.

Techniques: Activation Assay, Activity Assay, Transfection, Expressing, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Quantitative RT-PCR