Figure 1. GDP-exchange deficient ARF1 inhibits uptake of GPI-APs and the fluid-phase.

(a) IA2.2 cells (CHO cells expressing FR-GPI (FR) and human TfR) were transiently transfected with ARF1^T31N–GFP (outlined cells) for 18 h, and pulsed with Alexa⁵⁶⁸Mov19 Fabs and Alexa⁶⁴⁷ Tf (upper panels) or TMR–Dex (lower panels) for 10 min and processed for imaging. Images of internalized FR (red), TfR (green) and the fluid-phase (Fluid; red), are shown in grey-scale and colour merge. In transfected IA2.2 cells, the intracellular distribution of TfR containing perinuclear recycling compartment (REC) is altered, but Tf-uptake is unaffected. (b) MEFs transfected with ARF1^T31N–GFP for 20 h, were pulsed with labelled probes for 5 min, fixed and imaged on a confocal microscope. Grey-scale and colour merge images of internalized Cy5–CTxB (CTx; red) with TMR–Dex (lower panel; green) or Alexa⁵⁶⁸Tf (upper panel; green) from a single confocal section are shown (transfected cells are outlined). In transfected cells, uptake of CTxB is blocked and fluid uptake is significantly reduced while TfR-uptake is unaffected. (c) Histogram showing uptake of TfR, FR-GPI (normalized to surface receptor expression level) and fluid-phase in ARF1^T31N transfected cells plotted as a ratio to corresponding uptake measured in control cells. The error bars represent the weighted mean of fluorescence intensities ± s.e.m. (n = 61, 68, 100, 77, 63, 68; asterisks represent the cells transfected withARF1^T31N). The scale bars in a and b represent 10 μm.