split-neongreen tag ng2  (OpenCell Technologies Inc)

Journal: Contact

Article Title: OSBP is a Major Determinant of Golgi Phosphatidylinositol 4-Phosphate Homeostasis

doi: 10.1177/25152564241232196

Figure Lengend Snippet: Translocation of endogenous OSBP to mitochondrial outer membranes in response to ectopic PI4P synthesis. (A) Genomic tagging of OSBP with a split NeonGreen2 tag. (B) Representative confocal micrographs of parental HEK293A cells, NG2-OSBP knock in cells and NG2-OSBP knock in cells transfected for 48 hours with short interfering RNA against OSBP. DIC images were subject to a Fourier bandpass filter to correct for non-uniform illumination. NG2 channels are shown at identical excitation and gain settings. (C) Average intensity of cells like those shown in B from two experiments (measuring between 147 and 198 cells per condition from random fields), showing depletion of NG2 signal in NG2-OSBP cells by siOSBP. (D) Co-localization of NG2-OSBP signal with antibody staining against endogenous OSBP. Fields are representative of two experiments. (E) Experimental strategy using chemically-induced dimerization to recruit a PI4K to the mitochondrial outer membrane to synthesize PI4P, and the hypothesized recruitment of OSBP via its PI4P-binding PH domain. (F) Confocal time-lapse of a representative NG2-OSBP cells after mitochondrial PI4P synthesis is induced with rapamycin. Note the appearance of green puncta on the mitochondria marker by FRB-mito (magenta). (G) As F, except the catalytically inactive PI4KA mutant, D1957A is used and no green puncta appear at mitochondria. (H–I) Quantification of the fluorescence intensity of FKBP-PI4KAC1001 (H) and NG2-OSBP (I) at mitochondria after rapamycin-induced dimerization of FKBP with FRB-mito. Data are grand means ± s.e. of two (PI4KA) or three (D1957A) independent experiments quantifying 7–9 cells each. (J) Area under the curve calculation for the data presented in I showing significant recruitment of NG2-OSBP by PI4KA (WT) but not the D1957 mutant.

Article Snippet: To this end, we employed genomic tagging of OSBP using a split-NeonGreen tag (NG2, ), as recently demonstrated by the OpenCell project ( ).

Techniques: Translocation Assay, Knock-In, Transfection, Small Interfering RNA, Staining, Membrane, Binding Assay, Marker, Mutagenesis, Fluorescence

Journal: Contact

Article Title: OSBP is a Major Determinant of Golgi Phosphatidylinositol 4-Phosphate Homeostasis

doi: 10.1177/25152564241232196

Figure Lengend Snippet: Endogenous OSBP visits the PM at ER contact sites, but makes no measurable impact on PM PI4P turnover. (A) Hypothesized recruitment of OSBP to ER:PM contact sites through PM PI4P, and its inhibition by OSW-1. (B) TIRFM of NG2-OSBP cells shows numerous puncta at the PM; the kymograph at right shows the view along the dashed line over 30 seconds, showing puncta persist for several seconds. The graphs show the puncta full-width at half maximum and intensity (in photons), exhibiting values consistent with single molecules. Data are means ± 95% C.I. from 9 cells from a representative experiment. Trend lines fit a normal or log-normal distribution. (C) NG2-OSBP particles viewed in TIRFM visit the PM mainly at ER:PM contact sites. Images show co-localization of NG2-OSBP with mCherry-Sec61β (ER marker), -MAPPER (ER:PM contact site marker) or -clathrin light chain (negative control). The graphs shows the relative enrichment of puncta at these markers; pale symbols represent individual cell measurements (3–10 per experiment), whereas larger symbols represent means of each of three experiments. Both symbol types are color matched by experiment. Grand mean ± s.e. is also indicated. (D) OSBP does not measurably impact PM PI4P turnover after inhibiting the PM PI4K with A1. COS-7 cells expressing the EGFP-P4Mx2 PI4P biosensor were imaged in TIRFM and treated with the indicated compounds. The time course plot shows TIRFM fluorescence over time, and is the grand mean ± s.e. of three independent experiments (10–13 cells per experiment). Area under the curve calculation is shown for these data. Individual points show means of each experiment; grand mean ± s.e. is also shown. Results of a one-way ANOVA are indicated; P values above the data are from Sidak's multiple comparisons test relative to the DMSO control.

Article Snippet: To this end, we employed genomic tagging of OSBP using a split-NeonGreen tag (NG2, ), as recently demonstrated by the OpenCell project ( ).

Techniques: Inhibition, Marker, Negative Control, Expressing, Fluorescence, Control