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: ER membranes exhibit phase behavior at sites of organelle contact

doi: 10.1101/707505

Figure Lengend Snippet: Nuclear membranes become separated after LICV media treatment. A. Large volumes of ER lumen are present on the nucleus in swollen cells where the inner and outer nuclear membranes are separately visible, as indicated by the arrow. Left: ER membrane labeled with Sec61β-mNeonGreen; center: ER lumen labeled with KDEL-mRFP; Right: overlay. B. Nuclear pores are excluded from the regions of separated inner and outer nuclear membranes. Left: Nup50-mEmerald; right: Sec61β-mCherry; right: overlay. Cells were imaged at 37 C, 5% CO2. Scale bars are 10 microns.

Article Snippet: Sec61β-mNeongreen was created by replacing the mCherry fluorescent protein with the mNeonGreen fluorescent protein with the NEB HiFi DNA Assembly Master Mix(E2621), according to the manufacturer’s instructions . mCherry-ER-3 (KDEL-mCherry, vAddgene plasmid #55041), mEmerald-Nup50-N-10 (Nup50-mEmerald, Addgene plasmid #54210), mEmerald-PMP-C-10 (PMP-mEmerald, Addgene plasmid #54235), mCherry-Peroxisomes-2 (peroxisome lumen marker, Addgene plasmid #54520) were gifts from Michael Davidson. pEGFP-C1-ADRP (ADRP-GFP) was a gift from Elina Ikonen (Addgene plasmid #87161).

Techniques: Labeling