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: ORP3 was deleted from HeLa cells using CRISPR-Cas9 and several clones were characterized. A Immunofluorescence staining with polyclonal anti-ORP3 polyclonal antibodies in clonal cell lines from CRISPR Mock and ORP3-KO (3 distinct clones) HeLa cells (white). Nuclei were counterstained with DAPI. Bar: 10 µm. B Immunoblot staining for ORP3 from WT and CRISPR Mock or ORP3-KO (clones 1, 2) cells. GAPDH was used as a loading control. C In fixed cells stained with DAPI, ORP3-KO cells exhibited an accumulation of multinucleated cells. Clone 1 shown. Bar: 20 µm. D Statistical analysis of the fraction of multinucleated cells in WT, Mock (3 distinct clones) and ORP3-KO cells (5 distinct clones). Clone 1 (red circles), was used for the following experiments. E Live-cell imaging of mitosis from prophase to cytokinesis in WT HeLa cells (top) and ORP3-KO cells (lines a-d), showing typical ORP3-KO mitotic phenotypes: (a) no defect, (b) lagging chromosomes, (c) no DNA segregation between the two daughter cells and (d) segregation of all the DNA into only one of the daughter cells. DNA was stained with SiR-DNA. F Quantification of overall division defects in WT, Mock and ORP3 KO cell lines n=3 experiments. G Duration of cell division from nuclear envelope breakdown to the beginning of DNA decondensation in WT, Mock and ORP3-KO cells. Actual acquisition frequency was 3 minutes (10 minutes binning on the graph). N=3, 20-30 cells/experiment. H Immunofluorescence staining with anti ORP3 antibodies (white) and DAPI (blue) of WT, scramble siRNA- or ORP3 siRNA-treated HeLa cells. Bar: 10 µm. I Immunoblot staining for ORP3 from WT, scramble siRNA or ORP3 siRNA-treated (2 distinct siRNAs) HeLa cells. GAPDH was used as a loading control. J Quantification of the division defects in scramble and ORP3 siRNA treated HeLa cells. Light blue: siRNA/Scr1, dark blue: siRNA/Scr2 (n=6).

Article Snippet: Primary antibodies used for the Western-blotting: anti-ORP3 mouse IgG monoclonal antibody (1:1000 dilution, Santa Cruz Biotechnology, sc-398326); anti-VAPA rabbit polyclonal antibodies (1:1000 dilution, Atlas Antibodies, ref: HPA009174); anti-GAPDH mouse IgG monoclonal antibodies (1:2000 dilution, GeneTex, ref: GTX627408); anti α-tubulin mouse IgG monoclonal antibodies (1:1000 dilution, GeneTex, ref: GTX628802).

Techniques: CRISPR, Clone Assay, Immunofluorescence, Staining, Western Blot, Control, Live Cell Imaging

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 VAPA-immunoprecipitation of non-synchronized HeLa cells and cells synchronized by a double thymidine + nocodazole block (T+N). VAPA is detected in the input (left), and after VAP immunoprecipitation (VAPA IP) in both conditions (- or + T+N block). ORP3 is present in the input in comparable amounts but display a migration delay in synchronized cells (T+N). In T+N synchronized cells, ORP3 immunoprecipitation by VAPAis strongly enhanced. B In HeLa cells extracts from synchronized (T+N) or non-synchronized, treatment with the CIP phosphatase reversed the migration delay of ORP3 in T+N synchronized cells. C Scheme of ORP3 and the sequence of its FFAT-like domain (Core) and amino acids down-stream. Mass spectrometry has confirmed that many ORP3 residues were phosphorylated in synchronized cells (but not non-synchronized cells), including the three S and T residues of the FFAT-like domain and 3 serines just downstream of it (all highlighted in yellow). D Design of a FFAT-like phosphomimetic mutants, where S and T residues in the FFAT-like motif, which correspond to acidic residues in canonical FFAT, are replaced by aspartic acid (D). E WT-HeLa cells double-transfected with mCherry-KDEL and GFP, WT-GFP-ORP3 or the phoshomimetic mutant of ORP3-FFAT-like:(GFP-phospho-FFAT-like). Bar=20µm. F Quan-tification of the ER co-localization of GFP alone, GFP-WT-ORP3, and GFP-phosphomimetic mutant (P-FFAT-like). G VAPA-KO HeLa cells double-transfected with mCherry-KDEL and GFP, WT-GFP-ORP3 or the phoshomimetic mutant of ORP3-FFAT-like:(GFP-phospho-FFAT- like). Bar=20µm - H Quantification of the ER co-localization of GFP alone, GFP-WT-ORP3, and GFP-phosphomimetic mutant (P-FFAT-like) in VAPA-KO HeLa cells.

Article Snippet: Primary antibodies used for the Western-blotting: anti-ORP3 mouse IgG monoclonal antibody (1:1000 dilution, Santa Cruz Biotechnology, sc-398326); anti-VAPA rabbit polyclonal antibodies (1:1000 dilution, Atlas Antibodies, ref: HPA009174); anti-GAPDH mouse IgG monoclonal antibodies (1:2000 dilution, GeneTex, ref: GTX627408); anti α-tubulin mouse IgG monoclonal antibodies (1:1000 dilution, GeneTex, ref: GTX628802).

Techniques: Immunoprecipitation, Blocking Assay, Migration, Sequencing, Mass Spectrometry, Transfection, Mutagenesis

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 of stable GFP-ORP3 HeLa cells stained with SiR-DNA during mitosis. Top inset: in interphase, VAPA is mostly cytosolic. ORP3 is recruited to the ER from prophase to telophase, with a final enrichment at the midbody later in abscission (bottom inset). B localization of the ER-plasma membrane contact probe GFP-mapper in interphase (inset) and throughtout mitosis. Blue: Sir-DNA staining. C FFAT-dependent ER localization of ORP3. In live cell imaging, ORP3-KO HeLa cells stably expressing WT-GFP-ORP3 colocalizes with mCherry-VAPA at the ER. This localization persists if either FFAT (ORP3-FFAT m ) or FFAT- like (ORP3-FFAT-like m ) are inactivated, but not when both are defective. D Detailed scheme of ORP3 FFAT and FFAT-like domain sequence. E SBF-SEM electron microscopy of ORP3-KO HeLa cells demonstrates the existence of ER-plasma membrane contacts from metaphase to anaphase even in the absence of ORP3. F Anti-VAPA antibody staining in WT HeLa cells and stable VAPA-KO cell lines (white) DNA is stained with DAPI (blue). G Western blot of VAPA and VAPB of control and VAPA-KO HeLa cell lines. Only the VAPA signal is abolished in VAPA-KO cells. Tubuline was used as loading control. H VAPA-dependent ER localization of ORP3. Live-cell imaging in metaphase of stable GFP-ORP3 in WT-HeLa cells (left) and VAPA-KO HeLa cells (second column), VAPA-KO cells rescued with WT-VAPA (third column) or VAPA mutated in its FFAT binding domain (VAPA KD/MD). Bar=20µm.

Article Snippet: Primary antibodies used for the Western-blotting: anti-ORP3 mouse IgG monoclonal antibody (1:1000 dilution, Santa Cruz Biotechnology, sc-398326); anti-VAPA rabbit polyclonal antibodies (1:1000 dilution, Atlas Antibodies, ref: HPA009174); anti-GAPDH mouse IgG monoclonal antibodies (1:2000 dilution, GeneTex, ref: GTX627408); anti α-tubulin mouse IgG monoclonal antibodies (1:1000 dilution, GeneTex, ref: GTX628802).

Techniques: Live Cell Imaging, Staining, Clinical Proteomics, Membrane, Stable Transfection, Expressing, Sequencing, Electron Microscopy, Western Blot, Control, Binding Assay