Figure 2.
LD-associated APOE is exposed to the cytoplasm. (A) Cartoon schematic of the fluorescence protease protection (FPP) assay to test the topology of fluorescently tagged proteins in live cells. Cells are treated with 30 µM digitonin for 1 min, which selectively permeabilizes the plasma membrane but not the internal membranes. After permeabilization, cells are treated with 50 µg/ml proteinase K (PK), which enters the permeabilized plasma membrane and degrades all cytoplasmic-facing fluorophores (green). Because the ER membrane is not permeabilized, proteinase K does not enter into the ER lumen and ER lumen-facing fluorophores are retained (blue). (B) Representative confocal slices of FPP performed on primary cortical rat astrocytes (− OA) transiently transfected with APOE3-mEm, the ER marker TagBFP2-KDEL, and labeled for LDs with BODIPY 665/676. After digitonin permeabilization and PK treatment, APOE signal on the surface of LDs was lost, but the luminal ER marker fluorescence was retained. A Gaussian filter with a radius of 1 pixel was applied to all images to improve visibility for print. Scale bars: 10 µm (left), 5 µm for zoom (right). (C) Quantification of the FPP assay demonstrated in A and B. The fluorescence intensity of the indicated marker after PK treatment was divided by its fluorescence intensity just before PK treatment. For the “ER ratio,” the mean intensity of TagBFP2-KDEL within the entire cell was measured before and after PK treatment. For the “LD ratio,” the mean fluorescence intensity of the indicated LD protein (APOE, PLIN2, or LiveDrop) surrounding BODIPY 665/676-labeled LDs was measured before and after PK treatment. Ratios close to 1 indicate minimal loss of signal after proteinase K treatment, as observed with the ER marker TagBFP2-KDEL. Lower ratios indicate loss of fluorescence upon PK treatment. N = 8–18 cells per condition, collected from three independent experiments. *P < 0.05, **** P < 0.0001. Dig., 30 µM digitonin. PK, +50 µg/ml Proteinase K. P values were calculated via the Wilcoxon rank sum test and Bonferonni-corrected for multiple comparisons.

LD-associated APOE is exposed to the cytoplasm. (A) Cartoon schematic of the fluorescence protease protection (FPP) assay to test the topology of fluorescently tagged proteins in live cells. Cells are treated with 30 µM digitonin for 1 min, which selectively permeabilizes the plasma membrane but not the internal membranes. After permeabilization, cells are treated with 50 µg/ml proteinase K (PK), which enters the permeabilized plasma membrane and degrades all cytoplasmic-facing fluorophores (green). Because the ER membrane is not permeabilized, proteinase K does not enter into the ER lumen and ER lumen-facing fluorophores are retained (blue). (B) Representative confocal slices of FPP performed on primary cortical rat astrocytes (− OA) transiently transfected with APOE3-mEm, the ER marker TagBFP2-KDEL, and labeled for LDs with BODIPY 665/676. After digitonin permeabilization and PK treatment, APOE signal on the surface of LDs was lost, but the luminal ER marker fluorescence was retained. A Gaussian filter with a radius of 1 pixel was applied to all images to improve visibility for print. Scale bars: 10 µm (left), 5 µm for zoom (right). (C) Quantification of the FPP assay demonstrated in A and B. The fluorescence intensity of the indicated marker after PK treatment was divided by its fluorescence intensity just before PK treatment. For the “ER ratio,” the mean intensity of TagBFP2-KDEL within the entire cell was measured before and after PK treatment. For the “LD ratio,” the mean fluorescence intensity of the indicated LD protein (APOE, PLIN2, or LiveDrop) surrounding BODIPY 665/676-labeled LDs was measured before and after PK treatment. Ratios close to 1 indicate minimal loss of signal after proteinase K treatment, as observed with the ER marker TagBFP2-KDEL. Lower ratios indicate loss of fluorescence upon PK treatment. N = 8–18 cells per condition, collected from three independent experiments. *P < 0.05, **** P < 0.0001. Dig., 30 µM digitonin. PK, +50 µg/ml Proteinase K. P values were calculated via the Wilcoxon rank sum test and Bonferonni-corrected for multiple comparisons.

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