Journal: Nature Communications

Article Title: Omegasome-proximal PtdIns(4,5)P 2 couples F-actin mediated mitoaggregate disassembly with autophagosome formation during mitophagy

doi: 10.1038/s41467-019-08924-5

Figure Lengend Snippet: Synchronous formation between BATS and circular F-actin structures during mitoaggregate disassembly. a A MitoTracker DeepRed FM-stained HeLa cell co-expressing EBFP2-Parkin and hrGFP-BATS was illuminated with 635 nm (white circle) to locally activate Parkin-mediated mitophagy. b Selected frames from a time-lapse movie (Supplementary Movie ) on the cellular region undergoing mitophagy in a after 635 nm illumination. Scale bar: 10 μm. c Two examples of synchronous formation of BATS and circular F-actin structures near Parkin-labeled mitoaggregates. MitoTracker DeepRed FM-stained HeLa cells co-expressing EBFP2-Parkin, LifeAct-EGFP, and TagRFP-BATS were illuminated with 635 nm to locally activate Parkin-mediated mitophagy. Scale bar: 1 μm. d Newly formed circular F-actin (green lines) and BATS-positive structures (red lines) are indicated on thresholded images from a time-lapse movie (Supplementary Movie ). Circular F-actin and BATS structures formation overlapped greater than 80% in time as shown in the graph ( n = 20 from five independent experiments). e Mitoaggregate-associated BATS sites are decreased under CK666 treatment. Total BATS counts near Parkin-labeled mitochondria were calculated from 200 min time-lapse images with 2 min interval (mean ± S.D.; control n = 11, CK666 n = 11 biologically independent samples; *** p < 0.0001 as evaluated using two-tailed unpaired Student’s t -test)

Article Snippet: EBFP2-Parkin and TagRFP-Parkin were constructed by subcloning EBFP2 (Addgene, 14893) and TagRFP (Evrogen, FP141) into EYFP-Parkin (Addgene, 23955) respectively.

Techniques: Staining, Expressing, Labeling, Control, Two Tailed Test

Journal: Nature Communications

Article Title: Omegasome-proximal PtdIns(4,5)P 2 couples F-actin mediated mitoaggregate disassembly with autophagosome formation during mitophagy

doi: 10.1038/s41467-019-08924-5

Figure Lengend Snippet: Non-disassembled mitoaggregates harbored reduced autophagosome generation and displayed delayed turnover. a Diagram illustrating the principle underlying photoclustering experiments. After 635 nm illumination, CRY2-Ub become conjugated to damaged mitochondria through Parkin catalyzed ubiquitination. 488 nm illumination drives CRY2 oligomerization, which helps to maintain the clustering of damaged mitochondria and delays disassembly. For clarity, representative CRY2-Ub were drawn. b A MitoTracker DeepRed FM-stained HeLa cell co-expressing EBFP2-Parkin, CRY2-EGFP-Ub, and TPC2-TagRFP was illuminated with 635 nm to locally activate Parkin-mediated mitophagy. Selected frames of mitoaggregates of interest 40–340 min after mitophagy initiation are shown. During mitophagy, a portion of the mitoaggregates were 488-nm illuminated (as indicated by white dotted circles) at both 80 and 140 min. c The progression of mitophagy in b 340 min after mitochondrial damage. EGFP-quenched and TPC2-positive Parkin-labeled mitochondria represents mature autophagolysosomes (yellow arrows). EGFP-fluorescence retaining and TPC2-negative Parkin-labeled mitochondria represents ubiquitinated mitochondria outside of lysosomes (blue arrows). Photoclustered mitoaggregates are outlined with a blue-dotted line. d Diagram illustrating the experimental flow of photoclustering experiments in single cells. e A MitoTracker DeepRed FM-stained HeLa cell co-expressing EBFP2-Parkin, CRY2-TagRFP-Ub, and hrGFP-BATS was illuminated with 635 nm to activate Parkin-mediated mitophagy at two separate locations (white circles) within a single cell. f Selected frames from a time-lapse movie (Supplementary Movie ) on cellular region containing mitoaggregates in e 0–366 min after 635 nm illumination. 488 nm mediated clustering (green dotted circles) were triggered at both 77 and 154 min. The red arrows denote BATS-positive structures on the surfaces of photoclustered mitoaggregates. g Total number of BATS puncta in non 488-nm illuminated and 488-nm illuminated regions as shown in f . Scale bar: 10 μm

Article Snippet: EBFP2-Parkin and TagRFP-Parkin were constructed by subcloning EBFP2 (Addgene, 14893) and TagRFP (Evrogen, FP141) into EYFP-Parkin (Addgene, 23955) respectively.

Techniques: Ubiquitin Proteomics, Staining, Expressing, Labeling, Fluorescence

Journal: Current Genomics

Article Title: Increasing the Coding Potential of Genomes Through Alternative Splicing: The Case of PARK2 Gene

doi: 10.2174/1389202915666140426003342

Figure Lengend Snippet: List of commercially available antibodies against human, rat and mouse PARK2.

Article Snippet: NB600-1540 , Novus biologicals , 399-412 , H1, H2, H5-H7, H10-H12, H18, H20.

Techniques:

Journal: Autophagy

Article Title: The mitophagy receptor BNIP3 is critical for the regulation of metabolic homeostasis and mitochondrial function in the nucleus pulposus cells of the intervertebral disc

doi: 10.1080/15548627.2022.2162245

Figure Lengend Snippet: BNIP3 loss does not result in a compensatory increase in levels and colocalization of other mitophagy receptors. (A, B) Western blot and densitometric quantification of mitophagy receptors BNIP3L, BCL2L13, and FUNDC1 in ShCtrl, Sh Bnip3 #1, and DFP-treated NP cells. (C, D) Immunofluorescence staining BNIP3L and quantification of BNIP3L colocalizing with mitochondria, and (E, F) Immunofluorescence staining FUNDC1 and quantification of FUNDC1 colocalizing with mitochondria. (G, H) Western blot and densitometric quantification of canonical mitophagy pathway protein PRKN in ShCtrl, Sh Bnip3 , and DFP-treated NP cells. Western blot data represent six independent experiments. Colocalization BNIP3L and FUNDC1 with mitochondria was measured from Z-stack images of 100 cells per group. Scale bar: 15 and 4 μm. Statistical significance was determined using a t-test (D, F) or One-way ANOVA (B, H) with Sidaks’s post hoc test as appropriate.

Article Snippet: Primary NP cells from Sprague Dawley (Charles River, strain 400), prkn / park2 KO and WT (Envigo, HsdSage:LE- Park2 em1Sage ) and bnip3 KO-mitoQC and WT-mitoQC reporter mice were isolated and maintained in Dulbecco’s modified Eagle medium (DMEM; Sigma, D6046) and 10% FBS supplemented with antibiotics [ ].

Techniques: Western Blot, Immunofluorescence, Staining