Fig. 4.

Effect of colchicine on disulfide reduction of the folate-FRET reporter and FR recycling. (A–C) Disruption of microtubule network did not impede disulfide reduction of the folate-FRET reporter. KB cells were incubated with colchicine (10 μM) for 2 h at 37°C followed by a 0.5-h pulse with folate-FRET reporter (100 nM). After further incubation for 12 h in fresh medium, cells were imaged for BODIPY fluorescence (A) and FRET-induced fluorescence (B) (488/520 nm and 488/595 nm, respectively). (C) Overlay of A and B. (D–F) FR-containing endosomes do not merge with recycling compartments dispersed from the perinuclear region. Cells were treated for 2 h with folate-rhodamine to label the recycling center with the red dye, followed by rinsing with acidic buffer (pH 3.5) to remove surface-bound folate-rhodamine. Cells then were incubated with colchicine (10 μM) for 2 h to disrupt microtubules, followed by a 2-h treatment with folate-BODIPY to label newly internalized FR. Finally, cells were imaged for BODIPY (D) and rhodamine (E) fluorescence (488/520 nm and 543/595 nm, respectively). (F) Overlay of D and E. (Scale bar: 10 μm.)