ptdins 4 p  (Echelon Biosciences)

Journal: Journal of Lipid Research

Article Title: Phosphatidylserine transporters ORP5 and ORP8 control cholesterol trafficking from the plasma membrane to the endoplasmic reticulum

doi: 10.1016/j.jlr.2026.100989

Figure Lengend Snippet: PI(4,5)P 2 , but not PI4P, mediates cholesterol-induced PM recruitment of ORP5/8. A: Representative confocal images of HeLa cells treated with/without of 40 μg/ml Chol-CD for 30 min, followed by staining with ALOD4 (accessible pool of cholesterol) or immunofluorescence with antibody against PI4P or PI(4,5)P 2 . Scale bars = 10 μm for all images. Relative intensity was quantified from two independent experiments. ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001 (unpaired two-tailed Student's t test). B: Quantification of live-cell TIRF on sample overexpressing EGFP-EV, 2xP4M (PI4P), PHPLCδ (PI(4,5)P 2 ) upon 40 μg/ml Chol-CD loading from 3 independent experiments. Data points represent mean ± 95% CI. C: Schematics showing Pseudojanin (PJ) constructs, enzyme functions and the membrane targeted Lyn11-FRB-iRFP construct together with PJ construct before and after rapamycin treatment. D: Summarized data of EGFP-tagged ORP5, and ORP8 relative fold change when co-expressed with PJ constructs (PJ-Dead, PJ-Sac1, and PJ-INPP5E as indicated) and treated as indicated in HeLa cells. Data were quantified from three independent experiments. E: Representative TIRF and Epi images in HeLa cells overexpressing EFGP-tagged ORP5/8 or their PI(4,5)P 2 binding mutants (ORP5 R136Q and ORP8 R158C) with/without 40 μg/ml Chol-CD for 30 min. F: Relative TIRF/Epi intensity of cells shown in Figure 2E from two independent experiments. ∗∗, P < 0.01; ∗∗∗∗, P < 0.0001 (ordinary one-way ANOVA with Dunnett's multiple comparisons test, mean ± 95% CI) G: Relative fold change of live-cell TIRF for HeLa cells overexpressing EFGP-tagged ORP5/8 or their PI(4,5)P 2 binding mutants from three independent experiments. Data points represent mean ± 95% CI. PM, plasma membrane; ORP, OSBP-related protein; PI4P, phosphatidylinositol 4-phosphate; TIRF, total internal reflection fluorescence; Epi, epifluorescence; FRB, FKBP-rapamycin binding; PI(4,5)P 2 ,phosphatidylinositol 4,5-bisphosphate; Chol-CD, cholesterol—methyl-β-cyclodextrin complex.

Article Snippet: For PM PI4P/PI(4,5)P 2 detection, cells were fixed and blocked as described for the PS labeling protocol above, followed by 1 h incubation with purified PI4P antibody (Echelon Z-P004) or PI(4,5)P 2 antibody (Echelon Z-P045) and 45 min incubation with secondary antibody.

Techniques: Staining, Immunofluorescence, Two Tailed Test, Construct, Membrane, Binding Assay, Clinical Proteomics, Fluorescence

Journal: bioRxiv

Article Title: Lipid transfer by ORP3 is required for the regulation of PI4P and PI(4,5)P2 at the plasma membrane in mitosis

doi: 10.1101/2025.10.22.684039

Figure Lengend Snippet: A Live cell imaging spinning disk confocal microscopy of PI(4,5)P2 and DNA (using SiR DNA 670) at 3 different mitotic stages in WT and ORP3-KO cells stably expressing mCherry-PH-PLCẟ. Bar=20µm. PI(4,5)P2 intensity is represented by a heat-map LUT. B Quantification of PI(4,5)P2 intensity along the plasma membrane in each of the phases. Membrane length was normalized with position 0/100 corresponding to one pole and 25 (and 75) to one on the two ingression furrows. C Quantification of PI(4,5)P2 intensity at the furrow in cytokinesis compared to the poles. n=2 experiments, each dot corresponds to one cell. D Live cell imaging spinning disk confocal microscopy of PI4P (mCherry-P4MSiDM) during cytokinesis in WT HeLa cells, or cells expressing GFP-ORP3-ΔORD (green). E Live cell imaging spinning disk confocal microscopy of actin (Sir Actin) and DNA (Spy-DNA). Bar = 20µm. F-G Quantification of the actin signal intensity at the ingression furrow (F), at the poles (G).

Article Snippet: Primary antibodies used for immunofluorescence: anti-ORP3 rabbit IgG polyclonal antibody (1:300 dilution, Abcam, ref 224212); anti-PI4P mouse IgM monoclonal antibody (1:200 dilution, Echelon Biosciences, ref Z-P004) anti-PI(4,5)P2 mouse IgM monoclonal antibody (1:200 dilution, Echelon Biosciences, ref Z-P045).

Techniques: Live Cell Imaging, Confocal Microscopy, Stable Transfection, Expressing, Clinical Proteomics, Membrane

Journal: bioRxiv

Article Title: Lipid transfer by ORP3 is required for the regulation of PI4P and PI(4,5)P2 at the plasma membrane in mitosis

doi: 10.1101/2025.10.22.684039

Figure Lengend Snippet: In interphase (top panel), ORP3 is only weakly associated to the ER and only to PI(4,5)P2 rich plasma membrane domains. ORP3 interaction domains as well as the inter-conversion between PI4P and PI(4,5)P2 by phosphatases and kinases can be regulated by intracellular signaling. During mitosis (middle panel), the FFAT-like domain is phosphorylated and ER-association of ORP3 is strongly enhanced. Since plasma membrane PI(4,5)P2 levels are also increased the plasma membrane, association of ORP3 at ER-plasma membrane contacts is potentiated. Following ORP3 “priming” at contact sites, the regulation of PI4P and PI(4,5)P2 becomes highly dependent on local phosphatases and kinases. At the end of cytokinesis, ORP3 is associated to the cytoplasmic bridge. Phosphatases such as OCRL are activated in order to hydrolyze PI(4,5)P2 into PI4P and ORP3 function allows to drain and deplete PI4P from the plasma membrane at the cytoplasmic bridge.

Article Snippet: Primary antibodies used for immunofluorescence: anti-ORP3 rabbit IgG polyclonal antibody (1:300 dilution, Abcam, ref 224212); anti-PI4P mouse IgM monoclonal antibody (1:200 dilution, Echelon Biosciences, ref Z-P004) anti-PI(4,5)P2 mouse IgM monoclonal antibody (1:200 dilution, Echelon Biosciences, ref Z-P045).

Techniques: Clinical Proteomics, Membrane