Single event visualization of unconventional secretion of FGF2

Abstract

FGF2 is exported from cells by an unconventional secretory mechanism. Here, we directly visualized individual FGF2 membrane translocation events at the plasma membrane using live cell TIRF microscopy. This process was dependent on both PI(4,5)P₂-mediated recruitment of FGF2 at the inner leaflet and heparan sulfates capturing FGF2 at the outer plasma membrane leaflet. By simultaneous imaging of both FGF2 membrane recruitment and the appearance of FGF2 at the cell surface, we revealed the kinetics of FGF2 membrane translocation in living cells with an average duration of ∼200 ms. Furthermore, we directly demonstrated FGF2 oligomers at the inner leaflet of living cells with a FGF2 dimer being the most prominent species. We propose this dimer to represent a key intermediate in the formation of higher FGF2 oligomers that form membrane pores and put forward a kinetic model explaining the mechanism by which membrane-inserted FGF2 oligomers serve as dynamic translocation intermediates during unconventional secretion of FGF2.

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.

Figure 2.

Unconventional secretion of FGF2-GFP depends on cell surface heparan sulfate proteoglycans. (A) Individual FGF2-GFP particles at the inner plasma membrane leaflet were imaged based on GFP fluorescence using TIRF microscopy as described in the legend to Fig. 1 and in Materials and methods. This analysis included CHO-K1 WT cells (control; a), CHO-K1 WT cells treated with 50 mM NaClO₃ to inhibit post-translational sulfation (b), and CHO-745 mutant cells that express only the core protein without heparan sulfate chains (c). For each condition indicated, the first frame of TIRF videos is shown. Individual particles were identified using the TrackMate Fiji plugin (circles). Bar, 10 µm. (B) Quantification of FGF2-GFP particles at the inner plasma membrane leaflet under the conditions shown in A. TIRF videos of 300 frames (80 ms/frame) were analyzed using the plugin TrackMate (n > 70 cells per condition). The mean values of each condition are shown in brackets with FGF2-GFP set to 1. The statistical analysis was based on a one-way ANOVA test combined with Tukey’s post hoc test (ns, P ≥ 0.05; ***, P ≤ 0.001). (C) Individual FGF2-GFP particles on cell surfaces were imaged using fluorescent anti-GFP nanobodies. This analysis was done under the same experimental conditions shown in A. Cells were induced to express FGF2-GFP for 24 h followed by incubation with Alexa Fluor 647–labeled anti-GFP nanobodies for 30 min on ice. Cells were fixed, and images were acquired using both wide-field (GFP fluorescence) and TIRF microscopy (FGF2-GFP cell surface population). Bar, 10 µm. (D) Quantification of individual FGF2-GFP particles on cell surfaces under the conditions described in the legend to C. Data were processed using the Fiji plugin TrackMate (n > 144 cells per condition). For each condition, the mean value is shown in brackets with FGF2-GFP set to 1. The statistical analysis was based on a one-way ANOVA test combined with Tukey’s post hoc test (***, P ≤ 0.001).

Figure 3.

FGF2 membrane recruitment and translocation to cell surfaces depend on interactions of FGF2 with PI(4,5)P₂ at the inner leaflet. (A) CHO-K1 cells were imaged expressing FGF2-GFP at low levels to allow for single particle detection. Where indicated, cells were treated for 3 h with 10 mM neomycin. Single FGF2-GFP particles were identified at the inner plasma membrane leaflet (red circles). The first frames of TIRF videos are shown. Bar, 6 µm. (B) Quantification of FGF2-GFP recruitment at the inner leaflet of the plasma membrane under the conditions shown in A. TIRF videos of CHO-K1 cells expressing FGF2-GFP were acquired (300 frames; 80 ms/frame). The number of GFP particles at the inner plasma membrane leaflet was quantified using the Fiji plugin TrackMate (n > 95 cells per condition). The mean value of FGF2-GFP particles for each condition is given in brackets with the control set to 1. The statistical analysis was based on a one-way ANOVA test combined with Tukey’s post hoc test (***, P ≤ 0.001). (C) Identification of single FGF2-GFP particles at the outer plasma membrane leaflet. CHO-K1 cells expressing FGF2-GFP were cultivated in the presence or absence of 5 mM neomycin as indicated. Following incubation for 24 h in the presence of doxycycline to induce FGF2-GFP expression, intact cells were stained for FGF2-GFP on cell surfaces using anti-GFP nanobodies coupled to Alexa Fluor 647. Afterward, cells were fixed and imaged using both wide-field (green channel) and TIRF microscopy (red channel), the latter identifying single FGF2-GFP particles on cell surfaces. Bar, 6 µm. (D) Quantification of single FGF2-GFP particles on cell surfaces under the conditions shown in C including a titration of neomycin at 2, 5, and 10 mM. Raw data were analyzed using the Fiji plugin TrackMate (n > 108 cells per condition). The mean value of FGF2-GFP particles on cell surfaces for each condition is given in brackets with the control set to 1. The statistical analysis was based on a one-way ANOVA test combined with Tukey’s post hoc test (****, P ≤ 0.0001). (E) CHO-K1 cells were induced to express either FGF2-GFP, FGF2mt-GFP, a secretion deficient variant form of FGF2-GFP (C77/95A, Y81F; KRK128/129/133QQQ), or GFP at levels that allow for single-particle detection at the inner plasma membrane leaflet. Time series of 300 frames (80 ms/frame) were acquired, and for each condition, single particles were identified and quantified. The first frames of TIRF videos are displayed. (Bar, 6 µm). The mean value of FGF2-GFP particles at the inner leaflet for each condition is given in brackets with the control set to 1. The statistical analysis was based on a one-way ANOVA test combined with Tukey’s post hoc test (***, P ≤ 0.001). (F) CHO-K1 cells were induced for 24 h with doxycycline to express either FGF2-GFP, FGF2mt-GFP, a secretion-deficient variant form of FGF2-GFP (C77/95A, Y81F; KRK128/129/133QQQ), or GFP. Intact cells were stained for FGF2-GFP on cell surfaces using anti-GFP nanobodies coupled to Alexa Fluor 647. Afterward, cells were fixed and imaged using both wide-field (green channel) and TIRF microscopy (red channel), the latter identifying single FGF2-GFP particles on cell surfaces (bar, 6 µm). The number of single particles on cell surfaces was analyzed using the Fiji plugin TrackMate (n > 95 cells per condition). The mean value for each condition is displayed in brackets with the WT form of FGF2-GFP set to 1. The statistical analysis was based on a one-way ANOVA test combined with Tukey’s post hoc test (***, P ≤ 0.001).

Figure 4.

Direct visualization of individual events of FGF2 membrane translocation to the cell surface of living cells. (A) Representative example of a FGF2-GFP membrane recruitment and translocation event. TIRF imaging was performed on living cells expressing FGF2-GFP in the presence of Alexa Fluor 647–labeled anti-GFP nanobodies to detect extracellular FGF2-GFP. Successive frames (50 ms/frame) from a zoomed-in area of interest are shown. The time point of FGF2-GFP membrane recruitment was set to 0. Bar, 1 µm. (B) Bar histograms of the fluorescence intensity of the FGF2-GFP translocation event shown in A as a function of time. (C and D) A total of 95 FGF2-GFP translocation events were analyzed. The time intervals between the appearance of FGF2-GFP particles within the evanescent field and nanobody binding at the cell surface were calculated (t_(nb) − t_(GFP)). The distribution of observed time intervals was plotted. (E) FGF2-GFP translocation events from C and D were classified into three categories depending on the order of disappearance of GFP and Alexa Fluor 647 nanobody (nb) particles following completion of FGF2-GFP membrane translocation: (a) FGF2-GFP and Alexa Fluor 647 nanobody particles disappear simultaneously (gray), (b) GFP particles disappear before Alexa Fluor 647 nanobody particles (red), and (c) Alexa Fluor 647 nanobody particles disappear before GFP particles (green). (F) For each translocation event from C and D, the duration of GFP fluorescence remaining after nanobody disappearance in the observed position was calculated. The color code corresponds to what is shown in E. (G and H) Probability plots used to test whether one- or two-step exponential processes fit the time intervals measured between FGF2-GFP recruitment at the inner leaflet and the appearance of single FGF2-GFP molecules at the outer plasma membrane leaflet as detected by binding of anti-GFP nanobodies. Black dots correspond to measured data. The blue dots are the outcome of stochastic simulations with the hypothesized probability distribution for each case.

Figure 5.

Determination of the oligomeric size distribution of FGF2-GFP by single particle brightness analysis. (A and B) Representative histograms of the fluorescence intensity distribution from individual experiments. CHO-K1 WT and CHO-745 mutant cells expressing FGF2-sfGFP at low levels were imaged to allow for single particle detection. Single FGF2-sfGFP particles localizing at the plasma membrane were identified, and their fluorescence intensity was measured and plotted. Three independent experiments were conducted with each of them including the analysis of at least 30 cells. (C) Comparison of the oligomeric state of FGF2-sfGFP at the inner plasma membrane leaflet of CHO-K1 and CHO-745 cells. The plotted data represent mean values (± SD based on three independent experiments). (D) Calibration curve of sfGFP oligomeric standards. Correlation between the mean fluorescence intensity and the sfGFP copy number for standard oligomers with different subunit numbers. Mean values and SDs from two independent replicates are shown. The red line represents the linear fitting of the data (r² = 0.997). The slope of the curve corresponds to the theoretical fluorescence intensity value of a single sfGFP molecule. (E) Intensity distribution of FGF2-mGFP at the plasma membrane of CHO-745 cells from a representative experiment. Approximately 3,500 particles were analyzed. The resulting histograms were fitted with a sum of Gaussians to estimate the occurrence of monomers (orange), dimers (green), trimers (yellow), and tetramers (magenta). The area under each curve was used to calculate the percentage of occurrence of each oligomeric species. (F) Percentage of FGF2-mGFP monomers, dimers, trimers, and tetramers calculated after labeling correction from the averaged distributions of species from two different experiments. (G and H) Fluorescence intensity plots of two representative individual FGF2-mGFP particles in CHO-K1 and CHO-745 cells showing two sequential photobleaching steps. (I) A model proposing a two-step process of FGF2 membrane translocation with a slow component (FGF2 oligomerization and membrane insertion) and a subsequent fast component (FGF2 capturing at the outer leaflet mediated by heparan sulfates).