Figure 6.
Increased cellular levels of cholesterol positively modulate FGF2 recruitment at the inner plasma membrane leaflet in living cells, as well as PI(4,5)P2-dependent FGF2 translocation to cell surfaces. (A) FGF2-GFP recruitment efficiency under conditions of enriched cellular cholesterol levels. CHO K1 and U2OS cell lines stably expressing FGF2-GFP in a doxycycline-dependent manner were imaged by single molecule TIRF microscopy as described previously (Dimou et al., 2019; Legrand et al., 2020). Before imaging, cells were treated with Cholesterol:Methyl-β-Cyclodextrin (1:10 M ratio) complexes for 1 h in culture conditions. Single FGF2-GFP particles were identified at the inner plasma membrane leaflet (labeled by pink circles in subpanels a and c). For each condition, a widefield image and the first frame of the corresponding TIRF video are shown (subpanels a and c; Scale bar = 6 μm). Quantification of FGF2-GFP membrane recruitment at the inner leaflet of CHO K1 and U2OS cells is shown in subpanels b and d, respectively. Time-lapse TIRF movies with a total of 80 frames (100 ms/frame) were analyzed using the Fiji plugin TrackMate (Tinevez et al., 2017). The number of GFP particles was normalized for both surface area and the relative expression levels of FGF2-GFP for each experimental condition. Mean values are shown in brackets, with the mock condition set to 1. Data are shown as mean ± SD (n = 4). The statistical analysis was based on a t test (****P ≤ 0.0001). (B) FGF2-GFP translocation efficiency under conditions of enriched cellular cholesterol levels. CHO K1 and U2OS cells were induced with doxycycline for 24 h to express FGF2-GFP. Following treatment with Cholesterol:Methyl-β-Cyclodextrin (1:10 M ratio) complexes for 1 h in culture conditions, cells were incubated on ice for 30 min with Alexa-Fluor-647-labeled anti-GFP nanobodies. After labeling of FGF2-GFP on cell surfaces, cells were fixed with 4% PFA at room temperature for 20 min and imaged by single molecule TIRF microscopy as established previously (Dimou et al., 2019). For each condition, a widefield image and the corresponding TIRF image is shown (subpanels a and c; scale bar = 6 μm). Quantification of FGF2-GFP membrane translocation in CHO K1 and U2OS for all conditions indicated is shown in subpanel b and d, respectively. Single nanobody particles were analyzed using the Fiji plugin TrackMate (Tinevez et al., 2017). The number of nanobody particles detected per cell was normalized for the surface area of the corresponding cell. The mean values for each condition are shown in brackets, with the mock condition set to 1. Data are shown as mean ± SD (n = 4). The statistical analysis was based on a two-tailed unpaired t test (****P ≤ 0.0001). Data distribution was assumed to be normal, but this was not formally tested.

Increased cellular levels of cholesterol positively modulate FGF2 recruitment at the inner plasma membrane leaflet in living cells, as well as PI(4,5)P2-dependent FGF2 translocation to cell surfaces. (A) FGF2-GFP recruitment efficiency under conditions of enriched cellular cholesterol levels. CHO K1 and U2OS cell lines stably expressing FGF2-GFP in a doxycycline-dependent manner were imaged by single molecule TIRF microscopy as described previously (Dimou et al., 2019; Legrand et al., 2020). Before imaging, cells were treated with Cholesterol:Methyl-β-Cyclodextrin (1:10 M ratio) complexes for 1 h in culture conditions. Single FGF2-GFP particles were identified at the inner plasma membrane leaflet (labeled by pink circles in subpanels a and c). For each condition, a widefield image and the first frame of the corresponding TIRF video are shown (subpanels a and c; Scale bar = 6 μm). Quantification of FGF2-GFP membrane recruitment at the inner leaflet of CHO K1 and U2OS cells is shown in subpanels b and d, respectively. Time-lapse TIRF movies with a total of 80 frames (100 ms/frame) were analyzed using the Fiji plugin TrackMate (Tinevez et al., 2017). The number of GFP particles was normalized for both surface area and the relative expression levels of FGF2-GFP for each experimental condition. Mean values are shown in brackets, with the mock condition set to 1. Data are shown as mean ± SD (n = 4). The statistical analysis was based on a t test (****P ≤ 0.0001). (B) FGF2-GFP translocation efficiency under conditions of enriched cellular cholesterol levels. CHO K1 and U2OS cells were induced with doxycycline for 24 h to express FGF2-GFP. Following treatment with Cholesterol:Methyl-β-Cyclodextrin (1:10 M ratio) complexes for 1 h in culture conditions, cells were incubated on ice for 30 min with Alexa-Fluor-647-labeled anti-GFP nanobodies. After labeling of FGF2-GFP on cell surfaces, cells were fixed with 4% PFA at room temperature for 20 min and imaged by single molecule TIRF microscopy as established previously (Dimou et al., 2019). For each condition, a widefield image and the corresponding TIRF image is shown (subpanels a and c; scale bar = 6 μm). Quantification of FGF2-GFP membrane translocation in CHO K1 and U2OS for all conditions indicated is shown in subpanel b and d, respectively. Single nanobody particles were analyzed using the Fiji plugin TrackMate (Tinevez et al., 2017). The number of nanobody particles detected per cell was normalized for the surface area of the corresponding cell. The mean values for each condition are shown in brackets, with the mock condition set to 1. Data are shown as mean ± SD (n = 4). The statistical analysis was based on a two-tailed unpaired t test (****P ≤ 0.0001). Data distribution was assumed to be normal, but this was not formally tested.

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