GEEC Endocytosis in Drosophila Figure 6. Arf1and Cdc42 but not Rho, Rac1 or Arf6 are required for dynamin independent endocytosis in S2R+ cells. (A) Depletion of mRNA of the GTPases Arf1, Cdc42, Rho, Arf6 and Rac1 as verified by reverse-transcriptase PCR on cells treated with the corresponding dsRNAs (+) or control zeocin dsRNA (zeo) for 4 days (left panel). Normalized uptake of cells depleted for these GTPases for 4 days was then quantitated as before (Figure 2B). Values are represented as mean6s.e.m. from at least 100 cells per treatment in 2 independent experiments. (B) Fdex endosomes visualized at high resolution in control and Arf1 depleted cells. Bar graph shows the average endosomal intensity and number per cell, normalized to control cells (mean6s.e.m., measured from .400 endosomes from at least 15 cells in each case). (C) Normalized uptake of cells depleted for combinations of arf1, shi and zeo dsRNAs. Mean6s.e.m. values are from one representative experiment with n.200 cells in each condition. Asterix denote P,0.05 by Student’s t-test. Bar in B = 5 mm. doi:10.1371/journal.pone.0006768.g006 membranes during rounds of Arf1 activation at late ER and early Golgi compartments [46,47]. It follows that the membrane bound fraction of GFP-GBF1 shows extensive colocalization with Arf1 in these organelles [16,48]. Recently, activated Arf1 has also been detected in punctuate structures close to/at the plasma membrane by TIRF microscopy by using the Arf-binding domain (ABD) of ARHGAP10 fused to GFP as a specific sensor [10]. The specificity of ABD for activated Arf1 stems from the observation that in the evanescent plane, it colocalizes with Arf1 puncta and with nascent pinosomes, and this localization is altered upon overexpression of dominant-negative Arf1 or depletion of Arf1 by shRNA [10]. To examine the role of GBF1 in Arf1 activation near the plasma membrane, cells expressing low levels of GFP-GBF1 were imaged with live TIRF microscopy. A fraction of the total (wide-field) pool of GFP-GBF1 was detectable as punctuate structures in the evanescent field (Figure 8A). In live cells co-expressing ABD-RFP and GFP-GBF1, two colour fast confocal scanning near the plasma membrane revealed a sub-population of puncta which associated with both probes (Figure 8B). In time lapse TIRF microscopy, a fraction of GFP-GBF1 structures that were tracked over time were found to associate with activated Arf1 as masked by ABD-RFP (Figure 8C left panel). Notably, the overexpression of the activation mutant GFP-GBF1E794K caused a striking loss of ABD puncta, leaving only a flat haze in the evanescent field (Figure 8C right panel). This altered distribution of the ABD sensor was similar to that observed previously with overexpression of the activation-impaired Arf1T31N mutant, and of Arf1 shRNA [10]. Qualitatively, the residence time of GFP-GBF1 labeled structures in the evanescent plane was also much longer in the E794K mutant (Figure 8C), which is consistent with previous reports that describe the prolonged membrane association of this cycling-defective protein [46,47]. GBF1, in keeping with its established role in ER-Golgi transport, could affect endocytosis indirectly by modulating the PLoS ONE | www.plosone.org 10 August 2009 | Volume 4 | Issue 8 | e6768