Journal: EMBO Reports

Article Title: Peri-mitochondrial actin filaments inhibit Parkin assembly by disrupting ER-mitochondria contacts

doi: 10.1038/s44319-025-00561-y

Figure Lengend Snippet: ( A ) Western blot for VAP-B and myc-tag showing VAP-B expression in control and myc-VAP-B overexpressed U2OS cells. INF2 is used as loading control. Molecular weight in kDa. ( B ) Quantification of CCCP-induced actin polymerization in control and myc-VAP-B-overexpressing U2OS cells. Data with 65 cells (control) and 53 cells (overexpressing myc-VAP-B) from 3 independent runs. Error ± SEM. ( C ) Averaged traces showing mitochondrial calcium fold change (MitoGCamp6f) following histamine stimulation (at time 0) in control (ER-RFP expressing), ER-RFP-mito (ER-Mito linker) and PM-RFP-Mito (PM-Mito-linker) transfected HeLa cells. Data from 2 independent experiments comprising of 4 individual traces for each condition. Error: ±SEM. ( D ) Dot plot of peak fold change in mitochondrial calcium following histamine stimulation from individual traces as in Fig S4C. Data for 32 cells (Control- ER-RFP); 32 cells (ER-RFP-Mito); 28 cells (PM-RFP-Mito) for 3 independent runs. Error: ±s.d. P = 0.0213 (*) for Control Vs ER-RFP-Mito, P = 0.0439 (*) for Control Vs PM-RFP-Mito and P = 0.0001 (****) for ER-RFP-Mito Vs PM-RFP-Mito, One-way ANOVA used. ( E ) Graph showing actin filaments intensity (GFP-Ftractin) from HeLa cells transiently expressing either ER-RFP-Mito (magenta) or PM-RFP-Mito or ER-RFP, treated with 20 µM CCCP. Data from 28 cells (ER-RFP); 27 cells (ER-RFP-Mito) and 23 cells (PM-RFP-Mito) from 4 independent experiments. Error: ±SEM.

Article Snippet: Primary antibody: anti-myc-tag antibody (Abcam, ab32 used 1:1000) anti-INF2 antibody (CST #41081; used at 1:1000) anti-VAP-B antibody (Proteintech #14477-1-AP; used at 1:5000 at RT) anti-LC3B antibody (CST #12741S; used at 1:1000) anti-Parkin antibody (CST #2132S; used at 1:1000) anti-Parkin(phospho S65) antibody (Abcam #315376; used at 1:1000) anti-Pink1 antibody (CST #6946S; used at 1:1000) anti-β-actin antibody (CST #3700S; used at 1:10,000) anti-Mfn2 antibody (CST #9482S; used at 1:1000) anti-VDAC antibody (Invitrogen #MA5-41088; used at 1:1000) anti-Phospho ubiquitin (Ser65) (CST #62802S; used at 1:1000).

Techniques: Western Blot, Expressing, Control, Molecular Weight, Transfection

Journal: bioRxiv

Article Title: Spinal cord phosphoproteome of a SCA2/ALS13 mouse model reveals alteration of ATXN2-N-term SH3-actin interactome and of autophagy via WNK1-MYO6-OPTN-SQSTM1

doi: 10.1101/2024.11.06.622233

Figure Lengend Snippet: (A) Schematic summary of phospho-regulation events of proteolysis and aggrephagy in the terminal-stage spinal cord of the Atxn2 -CAG100-KIN mouse model of SCA2/ALS13. The potential co-aggregation of ATXN2 (black clusters) with SH3-containing interactors and competing PRM-containing proteins that show differential phosphorylation is highlighted in a field with a gray background. Proteins potentially undergoing LLPS are highlighted in red letters, ALS disease proteins in bold letters. (B) Schematic protein structures and Q100-ATXN2-triggered differential phosphorylation events for ATXN2 as aggregating disease protein and for SQSTM1 as the main selective aggrephagy receptor, illustrating that massive hyperphosphorylations are flanking the Q100-KIN mutation replacing murine residue Q156 (purple line) and the three PRMs (deep blue square, with mouse residue numbers) in the N-terminus of ATXN2, and are targeting the ZZ (light gray square) and LIR (deep green column) domains of SQSTM1. Hypophosphorylations across remaining ATXN2 likely reflect its reduced abundance upon polyQ expansion . The numbering of residues adheres to UniProt reference sequences, ignoring the unstable polyQ repeat KIN in ATXN2. Proteins are represented by their gene symbols, phosphorylated residue numbers are shown for serine (S), threonine (T) or tyrosine (Y). IDR = intrinsically disordered region, prone to phase separation; LIR = LC3-interacting region; KIR = Keap1-interacting region; PB1 = Phox and Bem1p; PtdIns3P = phosphatidylinositol 3-phosphate; RTK = receptor tyrosine kinase; TBS = TRAF6-binding sequence; UBA = ubiquitin-associated; ZZ = Zinc finger domain.

Article Snippet: The following primary antibodies were used: ATXN2 (Proteintech 21776–1-AP, 1:1000), BNIP3 (Proteintech 68091-1-Ig, 1:1000), CTSB (Abcam ab214428, 1:1000), LC3 A/B (Cell Sig-naling 4108, 1:1000), NBR1 (Cell Signaling 9891, 1:500), OPTN (Proteintech 10837-1-AP, 1:1000), PPIA (Proteintech 10720-1-AP, 1:1000), SQSTM1/p62 (Cell Signaling 39749, 1:1000), TAX1BP1 (Proteintech 14424-1-AP, 1:1000), UBQLN2 (Novus Biologicals NBP2-25164, 1:1000), WDFY3 (Proteintech 55009-1-AP, 1:1000), WNK1 (Invitrogen PA5-28382, 1:1000).

Techniques: Phospho-proteomics, Mutagenesis, Residue, Binding Assay, Sequencing, Ubiquitin Proteomics