DCA alters the dynamic spatial distribution of various lipids and EGFR in Caco-2 basal PM.

(A) Caco-2 cells ectopically expressing a GFP-tagged lipid-binding domain were treated with 1μM DCA for 5 minutes. Intact basolateral PM sheets of Caco-2 cells were then attached to EM grids and immunolabeled with 4.5nm gold nanoparticles conjugated to anti-GFP antibody. Gold particles were imaged via transmission EM (TEM) at 100,000X magnification. Spatial distribution of gold particles within a 1μm2 PM area was quantified using univariate K-function. Lmax values above 1 indicate statistically meaningful clustering, whereas Lmax values below 1 indicate spatial uniformity. (B) Number of gold particles within the same 1μm2 PM area was counted as an estimate of lipid level in the PM. (C and D) Caco-2 cells expressing either GFP-PASS (to specifically tag PA) or EGFR-GFP were exposed to 1μM DCA for various time points (0–60 minutes) before immunogold labeling. Extent of univariate spatial distribution (C) and number of gold labeling (D) of GFP-PASS or EGFR-GFP were calculated as described above. (E and F) Caco-2 cells ectopically expressing GFP-PASS or EGFR-GFP were exposed to increasing concentrations of DCA (0–30μM) for 5 minutes before immunogold labeling. Spatial distribution (E) and number of gold particles within a 1μm2 PM area (F) were calculated. Changes in EGFR monomer (G), dimer (H) and oligomer (I) populations without / with 1μM DCA were calculated using the spatial analysis data. At least 15 PM sheets from individual cells were analyzed in each condition. Statistical significance of the univariate clustering analysis (A, C and E) was evaluated via comparing our data against 1000 bootstrap tests, with * indicating pB, D and F), with * indicating pG-I) was evaluated using one-way ANOVA with * indicating p<0.05.