Figure 4. TGN is involved in intracellular trafficking of internalized ASN agglomerates within caveolin-1 and Rank-5 coated vesicles. ( A – C ) LCI images show ingested, far-red fluorescently-labeled ASN agglomerates (red) rapidly moving within the cell at 5.6 ± 1.88 μm/min. The transport occurs within a caveolin-1 lined vesicle as shown in mCherry-caveolin-1 (green pseudo-color)-transfected A549 cells. ( D – F ) Analogous images of A549, transfected with GFP-Rank-5 (green) show that at the cell surface the ASN agglomerates are not associated with Rank-5 (red arrow) and they do not move laterally; however, particles compartment acquires Rank-5 “lining” when the lateral movement commences at 5.9 ± 2.2 µm/min. ( G – I ) Structured illumination microscopy (SIM) of a fixed sample shows ASN associated with both caveolin-1 (white) and Rank-5 (green) in primary hAECs. ( J – L ) TGN granules (green) labeled with Alexa-488 wheat germ agglutinin (WGA) show TGN movement path coordinated but not overlapping with ASN agglomerates (red) in primary hAEC, and following somewhat different trajectories than TGN (green versus red lines) ( L ). Scale bars: ( A – F , J – L ): 2 μm, ( G – I ): 1 To visualize particle exocytosis in real-time, A549 cells and primary hAECs were studied using total internal reflection (TIRF) fluorescent microscopy. In TRIF (and TEM), we found the basolateral membrane of epithelial cells to adhere to coverslip in patches (adhesion patches) with the membrane detached from the coverslip in- between. Next, we set the illumination depth for TIRF to be extremely shallow (< 100 nm, by adjusting the angle of the excitation beam 26 ) so as to illuminate only the coverslip-attached portions of WGA-labeled basolateral plasma membrane 27 and leaving the non-adhered “pockets” of cell membrane and the cell interior in the dark 28–30 .