Figure 3. APOE targets LDs from the ER.... | Rockefeller University Press

Figure 3.

$APOE targets LDs from the ER. (A) Representative frames from fast Airyscan movies showing the localization of LD-associated APOE relative to the ER after 4 h of treatment with 400 µM OA in TRAE3-H cells. Cells were transfected with APOE3-mEm and the ER marker TagBFP2-KDEL and labeled for LDs with BODIPY 665/676. In the merged images, the ER is in magenta and APOE is in green. The yellow lines across the merged images indicate the line of pixels used to create the linescan graphs to the right of the images. In the linescan graphs, the relative fluorescence intensity of BODIPY 665/676-labeled LDs is in cyan, APOE3-mEm is green, and the ER is magenta. Two different localization patterns were observed: “half rings,” in which APOE partially covers the LD surface and colocalizes with the ER, and “full rings,” where APOE fully encloses the surface of the LD and only partially colocalizes with the ER. Scale bars, 500 nm. (B) Immunogold electron micrographs of endogenous APOE localization at membrane contact sites between the ER and LDs in TRAE3-H cells treated with 400 µM OA for 5 h. The blue arrow points to a direct membrane contact between the ER and an LD. Yellow arrows mark APOE localized to the cytoplasmic face of the ER membrane. The red arrow marks APOE localized to the cytoplasmic surface of the LD. Scale bars, 200 nm. (C) Representative frames from confocal FRAP movies of APOE3-mEm on the surface of BODIPY 665/676-labeled LDs in primary rat cortical astrocytes during an OA pulse (200 µM OA for 4 h) or an OA pulse-chase (200 µM OA for 4 h followed by 2 h chase in complete media—OA). APOE fluorescence was bleached at the 0 s timepoint. Scale bar, 1 µm. (D) Normalized intensity of APOE signal within the bleach ROI over time, with t = 0 s denoting the time at which APOE was bleached. The bold center line is the mean normalized intensity, and the upper and lower bounds of the ribbon represent ± standard deviation (SD). N = 28 cells per condition, collected from three independent experiments. (E) Comparison of the rate constant of recovery k between OA pulse and pulse-chase conditions. The rate constant was derived by fitting each recovery curve to the equation y = C (1 - e−kt). N = 28 cells per condition, collected from three independent experiments. * P < 0.05. (F) Comparison of the mobile fraction between OA pulse and pulse-chase conditions. The mobile fraction was derived by fitting each recovery curve to the equation y = C (1 - e−kt), where C is equal to the asymptote of the curve i.e., the mobile fraction. N = 28 cells per condition, collected from three independent experiments. **** P < 0.0001. P values were calculated via the Wilcoxon rank-sum test. (G) Schematic illustrating interpretation of the results of the FRAP experiment. When APOE on the LD is bleached during the OA pulse, it recovers very rapidly with a high mobile fraction. This indicates that bleached APOE on the LD is rapidly exchanged for unbleached APOE. After a short washout, LD-associated APOE recovers slowly or not at all, indicating that unbleached APOE molecules are unable to replace bleached ones on the LD. We hypothesize LD-APOE exchanges with APOE on the cytoplasmic face of the ER via membrane bridges during OA loading. These bridges are reduced or lost after OA washout, preventing exchange of APOE between LDs and the ER.$

APOE targets LDs from the ER. (A) Representative frames from fast Airyscan movies showing the localization of LD-associated APOE relative to the ER after 4 h of treatment with 400 µM OA in TRAE3-H cells. Cells were transfected with APOE3-mEm and the ER marker TagBFP2-KDEL and labeled for LDs with BODIPY 665/676. In the merged images, the ER is in magenta and APOE is in green. The yellow lines across the merged images indicate the line of pixels used to create the linescan graphs to the right of the images. In the linescan graphs, the relative fluorescence intensity of BODIPY 665/676-labeled LDs is in cyan, APOE3-mEm is green, and the ER is magenta. Two different localization patterns were observed: “half rings,” in which APOE partially covers the LD surface and colocalizes with the ER, and “full rings,” where APOE fully encloses the surface of the LD and only partially colocalizes with the ER. Scale bars, 500 nm. (B) Immunogold electron micrographs of endogenous APOE localization at membrane contact sites between the ER and LDs in TRAE3-H cells treated with 400 µM OA for 5 h. The blue arrow points to a direct membrane contact between the ER and an LD. Yellow arrows mark APOE localized to the cytoplasmic face of the ER membrane. The red arrow marks APOE localized to the cytoplasmic surface of the LD. Scale bars, 200 nm. (C) Representative frames from confocal FRAP movies of APOE3-mEm on the surface of BODIPY 665/676-labeled LDs in primary rat cortical astrocytes during an OA pulse (200 µM OA for 4 h) or an OA pulse-chase (200 µM OA for 4 h followed by 2 h chase in complete media—OA). APOE fluorescence was bleached at the 0 s timepoint. Scale bar, 1 µm. (D) Normalized intensity of APOE signal within the bleach ROI over time, with t = 0 s denoting the time at which APOE was bleached. The bold center line is the mean normalized intensity, and the upper and lower bounds of the ribbon represent ± standard deviation (SD). N = 28 cells per condition, collected from three independent experiments. (E) Comparison of the rate constant of recovery k between OA pulse and pulse-chase conditions. The rate constant was derived by fitting each recovery curve to the equation y = C (1 - e^−kt). N = 28 cells per condition, collected from three independent experiments. * P < 0.05. (F) Comparison of the mobile fraction between OA pulse and pulse-chase conditions. The mobile fraction was derived by fitting each recovery curve to the equation y = C (1 - e^−kt), where C is equal to the asymptote of the curve i.e., the mobile fraction. N = 28 cells per condition, collected from three independent experiments. **** P < 0.0001. P values were calculated via the Wilcoxon rank-sum test. (G) Schematic illustrating interpretation of the results of the FRAP experiment. When APOE on the LD is bleached during the OA pulse, it recovers very rapidly with a high mobile fraction. This indicates that bleached APOE on the LD is rapidly exchanged for unbleached APOE. After a short washout, LD-associated APOE recovers slowly or not at all, indicating that unbleached APOE molecules are unable to replace bleached ones on the LD. We hypothesize LD-APOE exchanges with APOE on the cytoplasmic face of the ER via membrane bridges during OA loading. These bridges are reduced or lost after OA washout, preventing exchange of APOE between LDs and the ER.