Figure 1.
Figure 1. Establishing an experimental system to visualize and quantify individual events of FGF2 membrane recruitment at the plasma membrane and translocation to the extracellular space. (A) Single particles of FGF2-GFP were imaged in the vicinity of the plasma membrane using TIRF microscopy. Stable CHO-K1 cell lines expressing either FGF2-GFP or GFP in a doxycycline-dependent manner were used to detect GFP particles at the inner leaflet of the plasma membrane. The first frame of a time-lapse TIRF video is depicted. GFP particles were identified (circles) and quantified using the Fiji plugin TrackMate. Bar, 6 µm. (B) Single-cell analysis correlating the number of GFP particles at the inner leaflet of the plasma membrane with relative expression levels. Time-lapse TIRF videos with a total of 100 frames (80 ms/frame) were analyzed. GFP particles were quantified using the Fiji plugin TrackMate. Relative expression levels were analyzed measuring total GFP fluorescence at the first frame of each image sequence. The number of GFP particles was expressed as particles per surface area (µm2) and plotted as a function of the relative expression level of the corresponding cell. A minimum of 50 cells was analyzed for each protein. (C) Quantitative comparison of FGF2-GFP versus GFP particles at the plasma membrane in living cells. TIRF videos with a total of 300 frames (80 ms/frame) were analyzed. Raw data were analyzed using the Fiji plugin TrackMate. The number of GFP particles were normalized per surface area and relative expression levels of the corresponding cell (n > 200 cells for each condition). Mean values of each condition are given in brackets. An unpaired t test was used for statistical analysis (****, P < 0.0001). (D) CHO-K1 cells were induced with doxycycline to express FGF2-GFP or GFP for 24 h and incubated on ice for 30 min with Alexa Fluor 647–labeled anti-GFP nanobodies. Following fixation of cells, secreted FGF2-GFP bound to HSPGs on cell surfaces was imaged using both wide-field and TIRF microscopy for GFP and Alexa Fluor 647, respectively. Bar, 10 µm. (E) Quantification of single particles of FGF2-GFP and GFP at the outer leaflet of the plasma membrane using fluorescent anti-GFP nanobodies as described in D. Single particles detected per cell (n > 190) were analyzed using the Fiji plugin TrackMate. The mean values of each condition are shown in brackets with FGF2-GFP set to 1. An unpaired t test was used for statistical analysis (****, P < 0.0001). (F) Cells were induced for 24 h to express FGF2-GFP and labeled with fluorescent anti-GFP nanobodies for 30 min on ice. TIRF videos were acquired with frames of 20 s. After 40 s of image acquisition, heparin (1 mg/ml) was added. Representative images from the beginning and the end of the acquired videos are shown. Bar, 6 µm. (G) Quantification of nanobodies detected per frame for videos acquired as described in F. Three videos were analyzed per condition using the TrackMate Fiji plugin. The dotted line indicates the time point of heparin addition. The plotted data represent mean values ± SEM. (H) CHO-K1 cells induced to express FGF2-GFP with doxycycline for 24 h were treated with heparin (1 mg/ml) for 10 min or left untreated as a control. Cells were labeled with fluorescent anti-GFP nanobodies and fixed. TIRF images were acquired and analyzed using TrackMate for quantification of FGF2-GFP on cell surfaces (n > 300 cells per condition). The mean values of each condition are shown in brackets with FGF2-GFP set to 1. An unpaired t test was used for statistical analysis (****, P < 0.0001). (I) CHO-K1 cells were cultivated under conditions of low levels of FGF2-GFP expression that allow for single particle detection based on GFP fluorescence. TIRF videos were acquired with frames of 20 s, and heparin (1 mg/ml) was added 40 s after the start of data acquisition. Representative images from the beginning and the end of the acquired videos are shown, depicting GFP particles at the inner leaflet of the plasma membrane (circles) before and after addition of heparin. Bar, 6 µm. (J) Quantification of GFP particles detected per frame for videos acquired as described in I. Three videos were analyzed per condition using the TrackMate Fiji plugin. The plotted data represent mean values ± SEM. (K) CHO-K1 cells were induced to express FGF2-GFP in low levels that allow for single particle detection. TIRF videos (80 ms/frame) were acquired before and after addition of heparin (1 mg/ml) for at least 10 min. The GFP particles at the inner plasma membrane leaflet were quantified using the Fiji plugin TrackMate. For each condition, 50 frames were analyzed (n > 200 cells per condition). The mean values of each condition are shown in brackets with FGF2-GFP set to 1. An unpaired t test was used for statistical analysis. ns, not significant.

Establishing an experimental system to visualize and quantify individual events of FGF2 membrane recruitment at the plasma membrane and translocation to the extracellular space. (A) Single particles of FGF2-GFP were imaged in the vicinity of the plasma membrane using TIRF microscopy. Stable CHO-K1 cell lines expressing either FGF2-GFP or GFP in a doxycycline-dependent manner were used to detect GFP particles at the inner leaflet of the plasma membrane. The first frame of a time-lapse TIRF video is depicted. GFP particles were identified (circles) and quantified using the Fiji plugin TrackMate. Bar, 6 µm. (B) Single-cell analysis correlating the number of GFP particles at the inner leaflet of the plasma membrane with relative expression levels. Time-lapse TIRF videos with a total of 100 frames (80 ms/frame) were analyzed. GFP particles were quantified using the Fiji plugin TrackMate. Relative expression levels were analyzed measuring total GFP fluorescence at the first frame of each image sequence. The number of GFP particles was expressed as particles per surface area (µm2) and plotted as a function of the relative expression level of the corresponding cell. A minimum of 50 cells was analyzed for each protein. (C) Quantitative comparison of FGF2-GFP versus GFP particles at the plasma membrane in living cells. TIRF videos with a total of 300 frames (80 ms/frame) were analyzed. Raw data were analyzed using the Fiji plugin TrackMate. The number of GFP particles were normalized per surface area and relative expression levels of the corresponding cell (n > 200 cells for each condition). Mean values of each condition are given in brackets. An unpaired t test was used for statistical analysis (****, P < 0.0001). (D) CHO-K1 cells were induced with doxycycline to express FGF2-GFP or GFP for 24 h and incubated on ice for 30 min with Alexa Fluor 647–labeled anti-GFP nanobodies. Following fixation of cells, secreted FGF2-GFP bound to HSPGs on cell surfaces was imaged using both wide-field and TIRF microscopy for GFP and Alexa Fluor 647, respectively. Bar, 10 µm. (E) Quantification of single particles of FGF2-GFP and GFP at the outer leaflet of the plasma membrane using fluorescent anti-GFP nanobodies as described in D. Single particles detected per cell (n > 190) were analyzed using the Fiji plugin TrackMate. The mean values of each condition are shown in brackets with FGF2-GFP set to 1. An unpaired t test was used for statistical analysis (****, P < 0.0001). (F) Cells were induced for 24 h to express FGF2-GFP and labeled with fluorescent anti-GFP nanobodies for 30 min on ice. TIRF videos were acquired with frames of 20 s. After 40 s of image acquisition, heparin (1 mg/ml) was added. Representative images from the beginning and the end of the acquired videos are shown. Bar, 6 µm. (G) Quantification of nanobodies detected per frame for videos acquired as described in F. Three videos were analyzed per condition using the TrackMate Fiji plugin. The dotted line indicates the time point of heparin addition. The plotted data represent mean values ± SEM. (H) CHO-K1 cells induced to express FGF2-GFP with doxycycline for 24 h were treated with heparin (1 mg/ml) for 10 min or left untreated as a control. Cells were labeled with fluorescent anti-GFP nanobodies and fixed. TIRF images were acquired and analyzed using TrackMate for quantification of FGF2-GFP on cell surfaces (n > 300 cells per condition). The mean values of each condition are shown in brackets with FGF2-GFP set to 1. An unpaired t test was used for statistical analysis (****, P < 0.0001). (I) CHO-K1 cells were cultivated under conditions of low levels of FGF2-GFP expression that allow for single particle detection based on GFP fluorescence. TIRF videos were acquired with frames of 20 s, and heparin (1 mg/ml) was added 40 s after the start of data acquisition. Representative images from the beginning and the end of the acquired videos are shown, depicting GFP particles at the inner leaflet of the plasma membrane (circles) before and after addition of heparin. Bar, 6 µm. (J) Quantification of GFP particles detected per frame for videos acquired as described in I. Three videos were analyzed per condition using the TrackMate Fiji plugin. The plotted data represent mean values ± SEM. (K) CHO-K1 cells were induced to express FGF2-GFP in low levels that allow for single particle detection. TIRF videos (80 ms/frame) were acquired before and after addition of heparin (1 mg/ml) for at least 10 min. The GFP particles at the inner plasma membrane leaflet were quantified using the Fiji plugin TrackMate. For each condition, 50 frames were analyzed (n > 200 cells per condition). The mean values of each condition are shown in brackets with FGF2-GFP set to 1. An unpaired t test was used for statistical analysis. ns, not significant.

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