delivery, to the plasma membrane, of molecules critical to the process. We tested this possibility by first inhibiting Golgi-to-cell surface traffic with Brefeldin A (BFA), and then examining the effect of GBF1 activation on endocytosis. In this assay, wild type cells treated with BFA show increased levels of fluid phase uptake, presumably because factors required for this uptake are released from the Golgi compartment [10]. We found that GFP-GBF1 overexpression alone had no effect on increased fluid uptake following BFA treatment (Figure 8D). By contrast, expression of GFP-GBF1E794K caused a reversal of this BFA-dependent increase (Figure 8D). This reversal, also seen in cells expressing the Arf1T31N mutant [10], implicates BFA-insensitive GBF1 activation in the fluid uptake pathway. Together this data indicates a direct role for GBF1 in Arf1 activation in the vicinity of the plasma membrane. Discussion Conservation of the GEEC pathway in Drosophila This work demonstrates that the Arf1-mediated pathway in Drosophila bears a striking similarity to the GEEC pathway first described in mammalian cells [4,8,38]. In both cases, acidic early pinosomes which contain GPI-APs are segregated from transferrin cargo and from Rab5 marked endosomes. The formation of these pinosomes requires optimum levels of cholesterol, actin and a functional Arf1/Cdc42 machinery, and can proceed without functional dynamin and clathrin. Several clathrin-independent pathways other than the Arf1-mediated route have been described in mammalian cells - these depend on caveolin, RhoA, Arf6 or Rac1 [3,10]. The absence of caveolin in Drosophila and the lack of effect on endocytosis upon knockdown of Rho, Arf6 and Rac1 on the specific cargo tested indicate that these molecules may be recruited for specialized pathways that have recently emerged during mammalian evolution. The features of the Arf1-mediated GEEC pathway however, having been conserved across phyla, may represent the hallmarks of a primordial route of entry into metazoan cells. The present delineation of endocytic pathways in Drosophila S2R+ cells, coupled with previous evidence from primary cultures of hemocytes[19] and from S2 cells [39,49], argues for at least two routes of entry in Drosophila cells: a Tf and mBSA carrying pathway that requires clathrin and dynamin; and a bulk fluid phase uptake pathway that carries a large fraction of GFP-GPI. At present, we cannot rule out the presence of additional endocytic pathways, since a fraction of GFP-GPI is detected as separate from fluid cargo, and some Tf continues to enter cells in the absence of dynamin. The use of additional probes in combination with specific gene knockdowns will help to dissect these findings further. Following internalization, both receptor and fluid pathways converge onto Rab5 containing endosomes, where heterotypic and homotypic mixing occurs. The kinetics of mixing of transferrin/mBSA and fluid phase cargo in S2R+ cells are similar to those described for Drosophila hemocytes, where mixed cargo reached late endosomes within 5 min [19]. Compared to CHO cells, TfR recycling occurs at faster rates in S2R+ cells. While we have assumed, based on a parsimonious fit to the data that TfR efflux occurs as a first order process, a two component model that allows for fast and slow recycling steps can also be proposed [50]. A fast recycling step is consistent with the kinetics of Rab5 acquisition, which peaks at 2 min post-internalization and with the observed regurgitation of ,40% of internalized bulk fluid phase over 5 min. In this aspect, S2R+ cells, which are related to the macrophage S2 isolates [26], appear similar to specialized phagocytic cell types where fast receptor recycling is common GEEC Endocytosis in Drosophila [51,52]. While similarities exist between phagocytosis and macropinocytosis in mammalian cells [53] and molecules such as actin and Cdc42 are common to macropinocytosis and Arf1- mediated pinocytosis, the fluid phase pathway described here in Drosophila appears to be unstimulated and does not depend on Rac1 activity. We suggest that this pathway, akin to the GEEC pathway described in mammalian cells, is a distinct constitutive pinocytic process [4]. Our observations of differential, time-dependent sensitivity of fluid uptake to Latrunculin versus Jasplakinolide, as well as dependence of the process on Cdc42, suggest the requirement for a highly dynamic population of actin for mediating pinocytic entry. Recent evidence indicates that cortical actin dynamics drives the complexation of GPI-anchored proteins (GPI-APs) on the cell surface in a cholesterol dependent manner [9], and Cdc42 activity as well as the proper organization of GPI-APs is a requirement for internalization via the GEEC pathway [7,8]. Our description of a cholesterol-auxotrophic cell line defective in fluid phase uptake should allow further investigation of the relationship between cholesterol, GPI-AP organization and Cdc42 activity. Role of GBF1 in Arf1-mediated endocytosis There are several lines of evidence for the involvement of the GTPase exchange factor GBF1 in Arf1-mediated endocytosis. The depletion of the GBF1 ortholog garz in S2R+ cells results in specific inhibition of Arf1-dependent fluid uptake. Concurrently, expres- sion of the exchange defective GBF1E794K mutant results in the same defect in CHO cells. A fraction of GBF1 colocalizes with ABD-RFP - a specific sensor for activated Arf1- in the evanescent plane (,100 nm proximity to the plasma membrane). Notably, ABD puncta are lost from the evanescent plane upon expression of GBF1E794K, thus providing a direct link between GBF1 exchange activity, Arf1 activation at/close to the plasma membrane, and Arf1-mediated endocytosis. GBF1 is known to cycle rapidly between membranes and cytosol (t1/2,17 s; [46]). Membrane recruitment occurs via an unknown receptor(s) and is independent of Arf-GDP recruitment, while membrane dissociation occurs shortly after GTP exchange [46,48]. Sustained activation of Arf1 would require multiple rounds of GBF1 cycling and GTP exchange, as GBF1 cycles on/ off membranes faster than Arf1 [46]. As such, the fraction of GBF1 associated with activated Arf1 at any point in space and time would be relatively small. We were able to detect this small fraction in the evanescent field with the aid of sensitive cameras and low expression levels of GBF1 and ABD. Since the majority of membrane-associated GBF1 and ABD was located in pericen- triolar Golgi and transitional ER compartments (which rarely traverse the evanescent plane in TIRF imaging), good contrast was achievable near the plasma membrane where the concentration of GBF1 and ABD was relatively low. Since GBF1 was originally discovered as a GEF required for ER-Golgi transport in the secretory pathway [54], its role in endocytosis as ascribed here is novel. Extending an earlier finding that Arf1 activation continues to be required for endocytosis even after disruption of the early secretory pathway with BFA [10], we have shown here that the GEF activity of GBF1 is an additional requirement for this to occur. However, this raises the paradoxical issue of how GBF1 can still activate Arf1 in the presence of BFA, which is a non-competitive inhibitor that stabilizes a Arf-GDP- BFA-GEF complex, and causes morphological defects in vivo that mimic the loss of Arf1-GTP ([48] and refs. therein). As multiple GEFs can activate Arf1, and several are targeted by BFA [13], the routinely used concentrations of this drug may be sufficient to inhibit ER-Golgi traffic and disrupt Golgi complex morphology, PLoS ONE | www.plosone.org 13 August 2009 | Volume 4 | Issue 8 | e6768