Figure 1.
Quantification of Golgi apparatus and GM1 polarization.
(A) Golgi apparatus polarization was calculated as a vector (shown in red) that connects the center of mass of the nucleus (Cm nuc.) to the center of mass of the Golgi (Cm Golgi). From this Golgi vector, an angle is calculated with reference to the y axis, defined as 0° and facing the wound edge. When compared to the classical method, angles falling from −60° to +60° are the equivalent of an oriented Golgi falling within a 120° angle facing the wound edge (shaded area). (B) Plasma membrane polarization was determined by comparing the weighted fluorescence distribution of QDs in the plasma membrane with the geometric center of mass of the cell. GM1 gangliosides in the plasma membrane were labeled with cholera toxin subunit B conjugated to QDs, represented here as red circles. The weighted center of mass of GM1 QD fluorescence was calculated (Cm PM) along with the geometric center of mass of the cell (Cm cell). Subtracting the y component of Cm cell from the y component of Cm PM gives us ΔY, a measure of the shift in plasma membrane polarization towards the scratch.
Figure 2.
Golgi apparatus and GM1 polarization in response to LPA.
(A) Composite images (red, GM1; green, Golgi; blue, nuclei) from untreated wound-edge cells (none) and wound-edge cells incubated with LPA for 10 min or 30 min (scale bar, 10 µm). (B) Cumulative distributions of ΔY plasma membrane polarization values. At both 10 min and 30 min stimulation, ΔY values are significantly increased with respect to control. None, n = 121; 10 min, n = 152, p = 0.001; 30 min, n = 225, p = 0.001. (C) Cumulative distributions of the absolute values of Golgi angles. Compared to control, the LPA-stimulated Golgi apparatus is polarized after 30 min but not after 10 min. None, n = 111; 10 min, n = 150, p = 0.945; 30 min, n = 116, p = 0.001. *** represents p≤0.001 compared to control using the Kolmogorov-Smirnov test.
Figure 3.
Drug treatments reveal uncoupled pathways to Golgi apparatus and GM1 polarization.
(A) Composite images (red, GM1; green, Golgi; blue, nuclei) from control experiments (none), incubation with LPA for 30 min, and 30 min pretreatment with the drugs U0126, BFA, or Wortmannin before incubation with LPA (scale bar, 10 µm). (B) Comparison of cumulative distributions of ΔY plasma membrane polarization for all treatments. The plasma membrane is polarized in all cases compared to the unstimulated control, (none, n = 326; LPA, n = 323, p = 0.001; U0126, n = 87, p = 0.001; BFA, n = 104, p = 0.001; Wortmannin, n = 144, p = 0.003), but wortmannin incubation significantly inhibits polarization when compared to LPA incubation alone (LPA vs. Wort, p = 0.001). (C) Cumulative distributions of Golgi angles show that Golgi polarization is inhibited by BFA and U0126, but unaffected by wortmannin. None, n = 293; LPA, n = 322, p = 0.001; U0126, n = 87, p = 0.183; BFA, n = 104, p = 0.736; Wortmannin, n = 144, p = 0.001. *** represents p≤0.001 compared to control using the Kolmogorov-Smirnov test.
Figure 4.
Correlation between ΔY and the Golgi angle in individual cells.
The value of ΔY in µm and the absolute value of the Golgi angle are plotted on the y and x axes, respectively. One point corresponds to the value of ΔY and the Golgi angle calculated in a single cell. The Spearman's rank correlation coefficient ρ and associated p value are reported for each condition. None of the conditions has a significant correlation between GM1 polarization and Golgi apparatus polarization, and all slopes are near zero. None, n = 290, ρ = 0.01, p = 0.92; LPA 10 min, n = 150, ρ = −0.06, p = 0.44; LPA 30 min, n = 293, ρ = −0.08, p = 0.16; U0126, n = 87, ρ = 0.11, p = 0.32; BFA, n = 85, ρ = 0.06, p = 0.61; Wortmannin, n = 144, ρ = 0.01, p = 0.95.
Figure 5.
Analysis of GM1 distribution in response to drug treatments.
(A) GM1 gangliosides labeled with cholera toxin-QDot conjugates at a wound edge after exposure to LPA stimulation in the presence of drugs blocking either GM1 polarization or Golgi apparatus polarization (scale bar, 10 µm). (B) Total amount of GM1 labeling for each condition shown as frequency distributions. The GM1 labeling density is the percentage of total cell area covered by fluorescence signal based on thresholded particle analysis. Each point represents the value for a single cell. *** represents p≤0.001 compared to control using Kolmogorov-Smirnoff statistical tests. (C) Clustering behavior analysis. The average size of aggregates per cell in µm2 was plotted against GM1 labeling density, and Deming (model II) linear regression was performed to fit the data and determine slope. Slopes and the associated 95% confidence interval were used to determine significance between experimental groups. *** represents p≤0.001 compared to control using Student's t tests. For (B) and (C), none, n = 111; LPA 10 min, n = 151; LPA 30 min, n = 107; U0126, n = 86; BFA, n = 120; Wortmannin, n = 144.
Figure 6.
Cell migration wound closure assay after treatment with drugs.
(A) ECV304 cells grown to confluence and starved overnight were scratched with a pipette tip, and treated with either the continued presence of starvation medium (none), LPA stimulation, or LPA stimulation and inhibitory drugs U0126, wortmannin, wortmannin and U0126 combined, or BFA. Images of the scratch were collected after 0, 24 and 48 hours. (B) The wound area was measured at 3–5 points along the scratch for each time point and results were pooled for three experiments. Two-way ANOVA and Bonferroni post-tests comparing all columns were used to assess significance. P values and N, representing the number of frames collected and analyzed, are reported in Tables S1–S3 in File S1. Scale bar, 100 µm.
Figure 7.
Matrigel invasion assay in the presence of drugs.
ECV304 or LNCaP cells were counted and loaded into the upper chamber of a 0.8 µm pore insert containing matrigel basement membrane matrix. The lower chamber contained LPA chemoattractant, and both chambers contained inhibitors where indicated. Cells were allowed to migrate for 20 h before fixation and removal of unmigrated cells. Remaining migrated cells were stained to increase contrast, photographed and counted (A). The mean number of cells per frame was calculated from combined results from a minimum of 3 experiments (B) and (C). One-way ANOVA with Tukey post-tests to compare all columns was used to assess significance. The resulting p values and N, representing the number of frames used for analysis, are reported in Tables S4 and S5 in File S1. Scale bar, 100 µm.