Figure 6. Actin, myosin II, and Rab6A are engaged in the ASN-containing vesicle transport for exocytosis. TIRF microscopy at the basolateral side (illumination depth: < 250 nm). ( A–C ) GFP-Rab6A (green) marks the TGN and exocytotic vesicles and highlights the correlated movement of the TGN granule and with associated particle agglomerates (red). ( D – F ) Blebbistatin, a myosin II inhibitor, blocks the movement of the ASN compartments and the associated TGN. ( G ) The particles accumulate inside the cells in presence of blebbistatin. Data represent mean ± SD, n = 3. ( H ) Blebbistatin preincubation does not block the ASN uptake but their transport and exocytosis, since accumulated inside the pre-treated hAECs (Data are mean ± SD, n = 3 and **p < 0.01). ( I – K ) Labeled ASN- containing compartments (red) move along actin stress fibers (green) near the basolateral surface of A549 cells. The yellow track indicates the trajectory of a large agglomerate at the basolateral side. The agglomerates move along actin fibers at 4 microns during 50 s (Supplemental Movie S8). ( L ) Electron micrograph showing a particle ASN compartment adjacent to an F-actin bundle (black arrow) at the basolateral edge of the A549 cells. ( M ) A model of nanoparticle agglomerate transcytosis in lung epithelial cells. Formation of the uptake compart. Particles (red) adhere to the plasma membrane (dark blue) in a caveolin-dependent (cyan) way before the compartment forms ( I ). During internalization by actin (green) remodeling, the compartment remains associated with caveolin-1 and is cleaved off the plasma membrane by CtBP1 (magenta). At this point, Rank-5 (brown) is recruited to the compartment ( II ). The movement of the agglomerate compartment depends on Myosin II (pink). The compartment appears to be propelled by a trans-Golgi network (TGN) granule that moves along actin stress fibers. The TGN granule and the particle compartment are tethered to each other with a distance of up to 1 µm ( III ). Exocytosis of the particles after Rab6A is recruited to the TGN granule associated with the agglomerate-compartment. Rank-5 is removed at this point ( IV ). We have not directly observed the association of the TGN with its immersed actin network, myosin II and the actin stress-fiber and how this leads to the directed movement, nor is the nature of the tether known. The depiction is therefore based on the observation of function and is not literal. Scale bar ( A–C ) 2 μm, ( D – F ) 2 µm, ( L ) 200