1 Introduction [1] Trypanosoma brucei [2] [3] [2] T. brucei 2 Materials and methods 2.1 Disruption of TbARF1 expression by RNAi [2] T. brucei T. brucei [4] Not [5] 2.2 Microscopy [6] For transmission electron microscopy, cells were sequentially treated in 1% (w/v) glutaraldehyde for 1 h, 1% (w/v) tannic acid for 10 min, 0.5% (w/v) osmium tetroxide for 45 min (all in 100 mM phosphate buffer), then in 1% (w/v) aqueous uranyl acetate for 1 h. After dehydration in an acetone series, cells were embedded in Spurrs resin. Sections were cut on a Leica Ultracut, stained with saturated uranyl acetate in 50% ethanol and Reynolds lead citrate, and viewed with a Tecnai 12 BioTwin (FEI) at 120 kV. Images were acquired with a SIS MegaView III digital camera. 2.3 Trafficking assays 7 [7] 7 [8] 3 Results 3.1 TbARF1 is essential for viability in T. brucei procyclic cells [2] [9] Fig. 1 Fig. 1 3.2 Loss of ARF1 has no effect on fluid-phase endocytosis Fig. 2 Fig. 2 3.3 Loss of ARF1 causes an enlargement of the lysosome Fig. 2 Fig. 2 Fig. 2 [10] T. brucei [11] Fig. 2 3.4 Effects of ARF1 depletion on lysosomal trafficking and degradation of p67 [5,8] [8] Fig. 2 Fig. 2 4 Discussion [2] T. brucei [2] [12] [2] [13] [8] [13] T. brucei [14] T. brucei [2,15,16] T. brucei [17] [8] [8] [18] [8] [2] [2] Fig. 2