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Fig 1 | BMC Cell Biology

Fig 1

From: Dissecting seipin function: the localized accumulation of phosphatidic acid at ER/LD junctions in the absence of seipin is suppressed by Sei1pΔNterm only in combination with Ldb16p

Fig 1

Phosphatidic acid (PA) is concentrated in nuclear ER and lipid droplets in sei1Δ. a Opi1-GFP localization in wild type and in sei1Δ cells. Left, merged fluorescent and brightfield images are shown; equivalent intensity settings. Inset: contrast is enhanced in the area enclosed in dashed rectangle to more clearly show nuclear staining of Opi1-GFP in wild type cells. Right, percentage of cells with punctate staining. Average ± SE from three experiments, > 90 cells counted in each. b As in (A) but with Opi1-mCherry in W303 strain. c Immunoblot of Opi1-mCherry in wild type and sei1Δ cells, representative of three experiments. Signals were quantified using an Infrared detection system (Odyssey LiCoR Scanner), which showed that the Opi1-mCh signal was higher than that of wt (1.8 ± 0.2 fold). The cytoplasmic marker Zwf1p is used as a control. d Mutation of the FFAT motif in the chromosomal copy of Opi1-GFP reduces but does not eliminate the PA puncta. Left, GFP fluorescence; right, quantification of the percentage of cells with puncta. e Fluorescence microscopy of wild type and sei1Δ cells (W303 background) expressing GFP-Spo20(51–91). As in (A) but with GFP-Spo20(51–91). f Cells co-expressing Opi1-mCherry and Kar2-CFP-HDEL (ER marker) were stained with BODIPY 493/503. In the first merged images (fourth column), the BODIPY channel is green, Opi1-mCherry red, and Kar2-CFP-HDEL blue. The second merged images (fifth column) are magnifications of the preceding white boxes, illustrating Kar2 (ER, blue) and Opi1 (red). Scale bars, 5 μm., and all micrographs correspond to single optical sections. For quantification of cells with PA-puncta, at least 90 cells were counted in each of at least three independent experiments

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