phic31o coding sequence  (Addgene inc)

Journal: Nature

Article Title: A phosphoinositide signalling pathway mediates rapid lysosomal repair.

doi: 10.1038/s41586-022-05164-4

Figure Lengend Snippet: Fig. 2 | PtdIns4P drives ORP-tethered ER–lysosomal contacts for rapid membrane repair. a, ORP family members are enriched on damaged lysosomes. Note that ORP10 is absent from S20 (supernatant after centrifugation at 20,000 g) (arrow head). Asterisk indicates a nonspecific band. b, Endogenous ORP9 is recruited to LAMP1-positive lysosomes upon brief LLOME treatment in U2OS cells. c, Punctate intensities of endogenous ORP9 and ORP11 before and after LLOME treatment. More than 30 random cells were quantified for each condition. Data are mean ± s.e.m.; n = 4 trials. See fluorescence images in Extended Data Fig. 5m. d, LLOME induces extensive wrapping of endogenous LAMP1 by eGFP–VAPA in U2OS cells. Right, schematic illustration of the process. e, Percentage of U2OS cells with more than five lysosomes extensively wrapped by eGFP–VAPA. More than 100 cells were counted for each condition. Data are mean ± s.e.m.; n = 3 per condition. See fluorescence images in Extended Data Fig. 6a. f, Representative microscopy and quantification of ER–lysosome wrapping in various ORP-knockout cells. More than 100 cells were counted for each condition. Data are mean ± s.e.m.; n = 3. See more details in Extended Data Fig. 6c–f.

Article Snippet: To generate the Lyso-TurboID construct (pCDH-LAMP1-mGFP-TurboID), the LAMP1– mGFP sequence was amplified from Addgene Plasmid 34831 and fused with TurboID sequence into pCDH-CMV-MCS.

Techniques: Membrane, Centrifugation, Fluorescence, Microscopy, Knock-Out

Journal: Nature

Article Title: A phosphoinositide signalling pathway mediates rapid lysosomal repair.

doi: 10.1038/s41586-022-05164-4

Figure Lengend Snippet: Fig. 3 | ORP9, ORP10 and ORP11 mediate rapid lysosomal repair by ER-to-lysosome transfer of phosphatidylserine. a, Schematic of the in vitro lipid transport assay for ORP9 and ORP11. Note that nitrobenzoxadiazole (NBD) is attached to one fatty acid chain but not the head group of PS. PE, phosphatidylethanolamine; Rh, rhodamine; DGS–NTA(Ni), a nickel-chelating lipid that binds His-tagged proteins. b, Lipid transfer assay demonstrating the increase in NBD fluorescence stimulated by ORP9 and ORP11 in the presence of NBD–PS but not NBD–cholesterol (Chol) or TopFluor (TF)-cholesterol. Data are mean ± s.e.m.; n = 3 per condition. c, U2OS cells stably expressing the PS probe GFP–Lact-C2 were treated with LLOME for 20 min and washed with detergent before immunostaining of endogenous IST1 and LAMP1. Scale bars, 10 μm. d, Colocalization of GFP–Lact-C2 and LAMP1 in the indicated U2OS cell lines. Data are mean ± s.e.m. of Pearson’s correlation coefficient; n = 30 cells from 3 trials per condition. See fluorescence images in Extended Data Fig. 7f. e, Schematic illustration of different ORP mutants used in f,h. f, U2OS cells stably expressing GFP–Lact-C2 and indicated mCherry–ORP9 and ORP11 proteins (mutants are notated as ORP9 mutant/ORP11 mutant) were treated as in c, and the Pearson’s

Article Snippet: To generate the Lyso-TurboID construct (pCDH-LAMP1-mGFP-TurboID), the LAMP1– mGFP sequence was amplified from Addgene Plasmid 34831 and fused with TurboID sequence into pCDH-CMV-MCS.

Techniques: In Vitro, Transport Assay, Fluorescence, Stable Transfection, Expressing, Immunostaining, Mutagenesis

Journal: Nature

Article Title: A phosphoinositide signalling pathway mediates rapid lysosomal repair.

doi: 10.1038/s41586-022-05164-4

Figure Lengend Snippet: Fig. 4 | PS-activated lipid transport by ATG2 mediates direct, rapid lysosomal repair. a, Live-cell imaging demonstrating rapid recruitment of eGFP–ATG2A to damaged lysosomes in U2OS cells stably expressing eGFP– ATG2A and LAMP1–mCherry. Scale bar, 10 μm. b, Quantification of eGFP–ATG2A puncta in individual U2OS cells upon LLOME treatment. c, eGFP–galectin-3 assay showing defects of rapid lysosomal repair in four independent clones of ATG2A/B-DKO cells. Data are mean ± s.e.m.; 50–100 cells, n = 3 for each condition. See more details in Extended Data Fig. 9a,b. d, Experimental design for the reconstitution of PS-stimulated lipid transport by ATG2A using a FRET-based assay. e, PS in acceptor liposomes potently stimulates ATG2A-dependent NBD fluorescence. Data are mean ± s.e.m.; n = 3 per condition. f, Illustration of lipid transport mutants of ATG2A. Flexible loops outside of the main structure are not shown. g, In vitro lipid transport assays testing the activity of various ATG2A mutants. Data are mean ± s.e.m.; n = 3 per condition. h, eGFP–galectin-3 assay demonstrating that ATG2A lipid transport

Article Snippet: To generate the Lyso-TurboID construct (pCDH-LAMP1-mGFP-TurboID), the LAMP1– mGFP sequence was amplified from Addgene Plasmid 34831 and fused with TurboID sequence into pCDH-CMV-MCS.

Techniques: Live Cell Imaging, Stable Transfection, Expressing, Clone Assay, Liposomes, Fluorescence, In Vitro, Activity Assay