Fig. 1. BFA induces apical redistribution of TfR. 3-D stereopair images of living, polarized PTR cells imaged in the presence of apical Alexa488-Tf and basolateral Alexa568-Tf after 30 minutes of uptake, following 15 minutes of pretreatment in medium lacking (A) or containing (B) 10 µM BFA, show that BFA significantly increases apical Tf uptake. (Note that these and all subsequent 3-D stereopair images are also presented as rotating animations at http://renal.nephrology.iupui.edu/wangetal2). Note that the background fluorescence in these and subsequent images of living cells results from the presence of fluorescent ligands in the medium. (C-E) The speed with which BFA induces apical redistribution of TfR is shown in extended focus projected volumes of living cells preincubated with apical Alexa488-Tf and basolateral Alexa568-Tf for 20 minutes, and then in probes plus BFA for the indicated times. (F-H) Uptake of Tf from the apical chamber does not result from leakage of dye across disrupted tight junctions. (F) Vertical cross section of an image volume collected of control cells incubated with basolateral 70 kDa fluorescein dextran for 20 minutes show that the dye is limited to the basolateral medium. (G) The tight junction barrier is likewise intact in cells pretreated with 10 µM BFA, then in dextran and BFA for another 30 minutes. (H) The junction is breached in cells pre-incubated in PBS lacking Ca²⁺ for 30 minutes, then incubated with dextran in PBS for 20 minutes, resulting in leakage of dextran into the apical space. Scale bar, 10 µm for all panels except C-E (20 µm).