Journal: The Journal of cell biology

Article Title: PITPβ promotes COPI vesicle fission through lipid transfer and membrane contact formation.

doi: 10.1083/jcb.202407166

Figure Lengend Snippet: Figure 3. PITPβ promotes contact between the ER and COPI buds on the Golgi. Quantitative data are shown as mean ± SD, with the number of in- dependent experiments indicated. Statistics was performed using the two-tailed Student’s t test: ****P < 0.0001, ***P < 0.001, **P < 0.01, ns (non-significant) P > 0.05. (A) Colocalization of PITPβ with ER marker (Sec61β) and Golgi marker (giantin) as assessed by confocal microscopy using Airyscan, PITPβ (blue), Sec61β (green), giantin (magenta), n = 6. Representative images with scale bars are shown with inset highlighting PITPβ colocalizing with both Sec61β and Giantin (indicated by arrowheads). A reconstruction of this region is also shown, with a line scan along the dotted line providing quantitative information, as well as single-channel images. (B) Airyscan confocal microscopy examining the effect of siRNA against PITPβ on the colocalization of an ER marker (Sec61β) and a Golgi marker (Giantin). Quantitation of a representative experiment is shown, n = 3. (C) Airyscan confocal microscopy examining the effect of siRNA against PITPβ on the colocalization of an ER marker (Sec61β) and a TGN marker (TGN46). Quantitation of a representative experiment is shown, n = 3. (D) EM tomography showing COPI buds at the Golgi in close proximity to the ER membrane. A representative tomographic image slice is shown in the upper left panel with arrowheads pointing to COPI buds, bar = 200 nm. 3D reconstruction of the Golgi, COPI buds, and ER elements is shown in the lower left panel. Quantitation of a representative experiment is shown on the right, comparing the distance between COPI buds and ER membranes versus the distance between Golgi cisternal margins and ER membranes, n = 3. (E) Proximity ligation assay examining the effect of siRNA against PITPβ on the proximity between calnexin and giantin. Quantitation of a representative experiment is shown on right, n = 3. Representative confocal images are shown on left, PLA signal (red),

Article Snippet: VAP-A (104447) and VAP-B (104448) in pEGFP-C1 and mEmerald-tagged Sec61β in pEGFP-C1 (90992) were obtained from Addgene.

Techniques: Two Tailed Test, Marker, Confocal Microscopy, Quantitation Assay, Tomography, Membrane, Proximity Ligation Assay

Journal: bioRxiv

Article Title: PITPβ promotes COPI vesicle fission through lipid transfer and membrane contact formation

doi: 10.1101/2024.05.27.596058

Figure Lengend Snippet: Quantitative data are shown as mean ± s.e.m., with the number of independent experiments indicated. Statistics was performed using the two-tailed student t -test: **** p<0.0001, *** p<0.001, ns (non-significant) p>0.05. A , COPI transport assay assessing the effect of 20 µM H89 treatment. Quantitation is shown on right, n=3. Representative confocal images are shown on left, VSVG-KDELR (red) and giantin (green), bar=10 µm. B , Airyscan confocal microscopy examining the effect of siRNA against VAP-A on the colocalization of an ER marker (Sec61β) and a Golgi marker (giantin). Quantitation is shown on left, n=3. Representative images are shown on right, Sec61β (green), giantin (red), bar=10 µm. C , COPI transport assay assessing the effect of siRNA against VAP-A followed by functional lipid rescue by feeding cells with different lengths of PC. Representative confocal images are shown on left, VSVG-KDELR (red), giantin (green), bar=10 µm. Quantitation is shown on right, n=4. D , COPI transport assay assessing the effect of siRNA against VAP-A followed by functional lipid rescue by feeding cells with short forms of PE or PS. Quantitation is shown on right, n=3. Representative confocal images are shown on left, VSVG-KDELR (red) and giantin (green), bar=10 µm.

Article Snippet: VAP-A (104447) and VAP-B (104448) in pEGFP-C1 and mEmerald-tagged Sec61β in pEGFP-C1 (90992) were obtained from Addgene.

Techniques: Two Tailed Test, Transport Assay, Quantitation Assay, Confocal Microscopy, Marker, Functional Assay

Journal: bioRxiv

Article Title: PITPβ promotes COPI vesicle fission through lipid transfer and membrane contact formation

doi: 10.1101/2024.05.27.596058

Figure Lengend Snippet: Quantitative data are shown as mean ± s.e.m, with the number of independent experiments indicated. Statistics was performed using the two-tailed student t -test: **** p<0.0001, ** p<0.01, ns p>0.05. A , Colocalization of PITPβ with ER marker (Sec61β) and Golgi marker (giantin) as assessed by confocal microscopy using Airyscan, PITPβ (blue), Sec61β (green), giantin (red). A representative image is shown in upper left panel, bar=10 μm. An inset is shown in upper right panel with arrow heads highlighting PITPβ colocalizing with both Sec61β and Giantin. A reconstruction of the region highlighted by the arrow heads in the inset is shown in lower left panel. Line scan along the dotted line in the inset is shown in the lower right panel. B , The effect of siRNA against PITPβ on the colocalization of an ER marker (Sec61β) and a Golgi marker (giantin) as assessed by confocal microscopy using Airyscan. Quantitation is shown on right, n=6. Representative images are shown on left, bar=10 µm. Arrow heads highlight areas of colocalization. C , Airyscan confocal microscopy examining the effect of siRNA against PITPβ on the colocalization of an ER marker (Sec61β) and a TGN marker (TGN46). Representative images are shown on left, bar=10 µm. Arrow heads highlight areas of colocalization. Quantitation is shown on right, n=4. D , EM tomography analysis of COPI buds at the Golgi showing close proximity to ER membrane. A representative tomographic image slice is shown in upper left panel with arrow heads pointing to COPI buds, bar=200 nm. 3D reconstruction of the Golgi, COPI buds, and ER elements is shown in lower left panel. Quantitation is shown on right, comparing distance between COPI buds and ER membranes versus distance between Golgi cisternal margins and ER membranes, n=3. E , Proximity ligation assay examining the effect of siRNA against PITPβ on the proximity between calnexin and giantin, n=5. F , Proximity ligation assay assessing the effect of PITPβ depletion on the colocalization of calnexin and ζ-COP, n=5. G , Proximity ligation assay examining the effect of siRNA against PITPβ on the proximity between calnexin and TGN46, n=4.

Article Snippet: VAP-A (104447) and VAP-B (104448) in pEGFP-C1 and mEmerald-tagged Sec61β in pEGFP-C1 (90992) were obtained from Addgene.

Techniques: Two Tailed Test, Marker, Confocal Microscopy, Quantitation Assay, Tomography, Membrane, Proximity Ligation Assay