cdb tnl  (Native Antigen Inc)

Journal: bioRxiv

Article Title: VPS13C/PARK23 initiates lipid transfer and membrane remodeling for efficient lysosomal repair

doi: 10.1101/2025.10.23.684214

Figure Lengend Snippet: ( a ) Volcano plot showing enrichment of VPS13C, OSBP and various lysosomal damage markers on lysosomes affinity-purified from HeLa cells treated with LLOMe (500 μM, 30 min). Green , proteins enriched in both intact (–LLOMe) and damaged (+LLOMe) lysosomes; blue , proteins selectively enriched in intact lysosomes; magenta , proteins selectively enriched in damaged lysosomes. ( b ) Whole cell lysates (WC) and lysosomes purified from untreated and LLOMe-treated HeLa cells (500 μM, 30 min) were subjected to immunoblot analysis using antibodies against VPS13C, OSBP, LAMP1, PI4K2A, hIST1, Gal3/LEG3 and p62/SQSTM. ( c ) Quantification of the data in (b). Bars show normalized values relative to control. Data are means ± SD. n = 4 biological replicates. ( d ) Time-lapse fluorescence images of U2OS wildtype (WT) and PI4K2A-KO cells expressing VPS13C-mClover or OSBP-GFP ( green ) that were fed CF633-labelled 70kDa dextran ( magenta ) and treated with 1 mM LLOMe for the indicated time. Cells were imaged by spinning disk microscopy. Scale bar, 5 µm. ( e ) Time-course plotting VPS13C- and OSBP-positive puncta per 100 μm area in cells treated as in (d). Data are means ± SD (VPS13C: n = 31 cells for WT, 29 cells for PI4K2A-KO; OSBP: n = 35 cells for WT, 33 cells for PI4K2A-KO) from 2 independent experiments.

Article Snippet: Chemical reagents were used at the following concentrations, unless indicated otherwise: 1 mM L-leucyl-L-leucine O-methyl ester (LLOMe; Bachem, 4000725), 200 μM glycyl-L-phenylalanine 2-naphtylamide (GPN; Abcam, ab145914), 75 nM LysoTracker TM Red DND-99 (Thermo Fisher Scientific; L7528), 200 nM Apilimod (Sigma-Aldrich, SML2974), 1.7 μM CF®633 dextran 70,000 MW (Biotium, 80141), 100 μg/ml 3 μm Polybead® Amino Microspheres (Polysciences Inc., 17145-5), 100 mM pHrodo TM Red Succinimidylester (ThermoFisher, P36600) and 100 nM BDP-FL-DBCO (Broadpharm, BP-23473).

Techniques: Affinity Purification, Purification, Western Blot, Control, Fluorescence, Expressing, Microscopy

Journal: bioRxiv

Article Title: VPS13C/PARK23 initiates lipid transfer and membrane remodeling for efficient lysosomal repair

doi: 10.1101/2025.10.23.684214

Figure Lengend Snippet: ( a ) Schematic of the cellular uptake of 3 µm polystyrene microbeads functionalized with the pH-sensitive dye pHrodo SE. ( b ) Differential interference contrast (DIC) and fluorescence images of HeLa cells expressing GFP-tagged LAMP1 ( green ) that were fed pHrodo-microbeads ( magenta ) for 24 h. Cells were imaged by spinning disk microscopy. Scale bar, 10 µm. ( c ) Time-lapse DIC and fluorescence images of HeLa cells expressing Halo-tagged EqtSM ( cyan ), fed pHrodo-microbeads ( magenta ) and treated with 1 mM LLOMe for the indicated time. Cells were imaged by spinning disk microscopy. Scale bar, 10 µm. ( d ) Time-lapse fluorescence images of U2OS cells expressing OSBP-GFP or VPS13C-mClover ( green ) that were fed pHrodo-microbeads ( magenta ) and treated with 1 mM LLOMe for the indicated time. Line scans show overlap of OSBP or VPS13C and pHrodo signals along the path of arrow, with the position of microbeads marked by grey circles. Scale bar, 10 µm. ( e ) Fluorescence images of U2OS cells containing 3 µm polystyrene microbeads and expressing ATG2C-Halo, ATG2C V3563Q -Halo, SPG20-GFP or SPG20 8xV-GFP ( magenta ) were treated with 1 mM LLOMe for the indicated time, and then immunostained for hIST1 ( green ) and counter stained with DAPI ( blue ). Cells were imaged by DeltaVision microscopy. Scale bar, 10 µm. ( f ) Time-lapse fluorescence images of U2OS cells expressing mClover-tagged VPS13C or VPS13C V3563Q ( green ), fed pHrodo-microbeads ( magenta ) and treated with 1 mM LLOMe for the indicated time. Cells were imaged by spinning disk microscopy. Scale bar, 10 µm. ( g ) Time-lapse fluorescence images of U2OS cells expressing VPS13C-GFP or VPS13C V3563Q -GFP ( green ) and fed CF633-labelled 70kDa dextran ( magenta ) were incubated in isotonic medium (100% Opti-MEM) and then incubated for 5 min in hypotonic medium (1% Opti-MEM). Scale bar, 10 µm. ( h ) Live-cell fluorescence images of U2OS VPS13C-KO1 cells that were fed 3 µm polystyrene microbeads and then co-transfected with VAPA-mCherry ( magenta ) and VPS13C-mClover ( green ) as indicated. Scale bar, 10 µm. ( i ) Schematic of how conformational flexibility of its ATG2C domain-containing C -terminus may enable VPS13C to distinguish stressed from injured lysosomes. See text for further details. Illustration adapted from .

Article Snippet: Chemical reagents were used at the following concentrations, unless indicated otherwise: 1 mM L-leucyl-L-leucine O-methyl ester (LLOMe; Bachem, 4000725), 200 μM glycyl-L-phenylalanine 2-naphtylamide (GPN; Abcam, ab145914), 75 nM LysoTracker TM Red DND-99 (Thermo Fisher Scientific; L7528), 200 nM Apilimod (Sigma-Aldrich, SML2974), 1.7 μM CF®633 dextran 70,000 MW (Biotium, 80141), 100 μg/ml 3 μm Polybead® Amino Microspheres (Polysciences Inc., 17145-5), 100 mM pHrodo TM Red Succinimidylester (ThermoFisher, P36600) and 100 nM BDP-FL-DBCO (Broadpharm, BP-23473).

Techniques: Fluorescence, Expressing, Microscopy, Staining, Incubation, Transfection