Figure 6.

Perinuclear TFR and pIgA-R complexes show a clustered organization upon internalization from opposite PMs in the presence of BFA. (A) The organization of pIgA-R and TFR complexes cointernalized from opposite PMs in the presence of BFA was assayed at the perinuclear region (green rectangle) using FRET confocal microscopy. (B) Live-cell confocal FRET imaging was performed at the perinuclear region of polarized MDCK-PTR cells upon internalization of Cy3-Tfn and Alexa488-pIgA-R ligand from the basolateral and apical surfaces, respectively, in the presence of BFA. Pseudocolor images depict the pixel-by-pixel distribution of D/A (a) Acceptor (b), and E% (c) levels at the perinuclear region. Examples of selected ROIs are shown as white squares. Bar, 5 μm. (C) E%, D/A and Acceptor levels were calculated for a wide range of perinuclear ROIs. E% values were plotted as a function of Acceptor levels for D/A ≈ 1 (□) and for D/A ≈ 2 (▵). Trendlines are shown as visual helpers (D/A ≈ 1, dotted line; D/A ≈ 2, dashed line). E% is largely independent of Acceptor levels and increases with decreasing D/A ratios, suggesting that TFR and pIgA-R complexes show a clustered organization in perinuclear endosomes, independently of BFA and internalization protocols. (D) Perinuclear E% using cointernalization from basolateral PM in the absence of BFA (◇; Figure 4C) or from opposite surfaces in the presence of BFA (○) were plotted as a function of Acceptor levels for D/A ≈ 1. Trendlines are shown as visual helpers (−BFA Bas. PM inter., dotted line; +BFA Opp. PM inter., dashed line). These two data sets are not significantly different (p > 0.001) using an ANCOVA statistical analysis (Table 2).