cargo at the early endosomal compartment, followed by a segregation of certain membrane cargo (TfR and GPI-APs) towards a recycling fate, and lysosomal direction of the fluid- phase and ligands of the scavenger receptor. A dynamin- independent pinocytic pathway in S2R+ cells We next assessed if endocytic trafficking of any of the endocytosed cargo had a requirement for dynamin, one of a set of key molecular criteria used for the characterization of endocytic pathways [3]. S2R+ cells were treated with control dsRNA against the gene for zeocin resistance (which is absent in the Drosophila genome) or dsRNA corresponding to ,800 bp of coding sequence for the shibire gene, which codes for Drosophila dynamin. The dsRNA for shibire was effective in reducing protein levels for the target gene as seen in Western blots (Figure 4A). In control cells, TIRF imaging showed that antibodies to shibire label discrete puncta at the plasma membrane, which are presumably clathrin coated pits [19,43]. This localization was lost in shibire depleted cells (Figure 4A). Cells depleted for dynamin were pulsed with either Fdex or A647aGFP (to label GFP-GPI) and A568Tf for 3 min. The ratio of internalized aGFP or Tf to the surface levels of the respective receptors in each cell was determined quantitatively via MATLAB routines (see Figure S1 for methodology of quantitative endocytic assays). In cells without dynamin, the internalized to surface ratio of TfR was reduced ,50% compared to control cells (Figure 4B). The difference was largely due to an accumulation of TfR on the surface, probably reflecting a decreased rate of internalization of Tf. We verified this response to dynamin depletion using the fluorescent probe for the endogenous scavenger receptor, mBSA, in an accessibility assay (Figure 4C). The assay was used previously to assess the accessibility of clathrin coated pits to receptor ligands in Drosophila hemocytes harbouring a temperature sensitive shibire allele [19]. Here both control and shibire depleted cells showed similar quantitative levels of biotinylated mBSA (bmBSA) labeling, but the bmBSA in the shibire-depleted cells was ,2 fold more accessible to streptavidin (Figure 4C, indicative of surface-arrested receptor pits [19]. The effect of dynamin depletion on Tf endocytosis was similar to that of rapid dynamin inhibition with the small molecule inhibitor dynasore ([44]; Figure 4D). Notably, the levels of normalized GFP-GPI uptake were not different from controls in shibire-depleted (Figure 4B) or dynasore treated cells (Figure 4D). Similarly, these cells also exhibited normal or higher levels of Fdex uptake after loss of dynamin function (Figures 4B,D), and Fdex endosomes continued to accumulate in cells which contained surface-arrested bmBSA structures (Figure 4C). Thus, dynamin depletion in S2R+ cells inhibits internalization of two classes of receptors, and increases their surface accessibility, while GFP-GPI and fluid uptake is not inhibited. These data conclusively show that although TfR and scavenger receptors (for mBSA) are internalized using dynamin-dependent mecha- nisms, GPI-AP and fluid-phase uptake relies on mechanisms that do not utilize dynamin. Actin and cholesterol dependence of endocytic pathways Since agents that manipulate cholesterol levels in cells may have pleiotropic effects and can be toxic to the cells, the precise role of cholesterol in steady state cellular processes like constitutive endocytosis has been difficult to interpret. Here we exploit the finding that Drosophila cells can produce lipids from metabolic pathways, but lack key genes required for cholesterol biosynthesis [45]. By substituting normal serum with delipidated serum (which contains ,10 ug/ml cholesterol; see Materials and Methods) in fly GEEC Endocytosis in Drosophila growth medium, we could severely restrict the supply of cholesterol into S2R+ cells. We find that delipidated serum-adapted cells are able to grow well despite ,1% of normal sterol levels in their membranes (as measured by filipin binding; Figure 5A, and Carvalho et al., submitted). Notably, these adapted cells take in significantly lower levels of Fdex in a quantitative assay (Figure 5B). Furthermore, surface levels of TfRs are reduced in these cells (not shown), but their internalization is unaffected. The specific effect of cholesterol depletion on Fdex uptake can be reversed by incubating the depleted cells in cholesterol containing medium for 12 hrs (Figure 5B). Repleted cells exhibit normal levels of Fdex uptake, and have high levels of Tf internalization when normalized to surface pools of the receptor (Figure 5B). The surface levels of TfR remain low in these repleted cells (not shown), perhaps owing to slower recovery kinetics over the 12 hr incubation period. In S2R+ cells, both Fdex and Tf uptake were highly sensitive to the actin disrupting agent Latrunculin A (Lat) or Jasplakinolide (Jas). Cells were incubated with 1 mM Lat, or 4 mM Jas for a total of 30 min, in which time they were pulsed with Fdex or TfR in the presence of drug for different periods. Both Fdex and normalized Tf internalization was reduced .50% at short pulse times in Lat (1 min; Figure 5C). By contrast, treatment with Jas had little effect on Fdex uptake at short pulse times, while strongly inhibiting TfR uptake. At longer pulse times, Fdex accumulated in Lat treated cells, while Jas treated cells showed lower levels of Fdex uptake, and TfR uptake appeared to recover to steady state levels seen in untreated cells (Figure 5C). To test whether the accumulation of Fdex in Lat treated cells is due to a strong inhibition of a recycling pathway that facilitates regurgitation of fluid [8], we examined cells that were pre-pulsed with Fdex and then chased over different times in the presence of Lat. Notably, a large fraction of accumulated Fdex leaves the cell over short times (,5 min) in untreated cells, while the presence of Lat inhibits this efflux (Figure 5D). In summary, our results indicate that the main features of a cholesterol and actin sensitive GPI-AP endocytic pathway that does not utilize Dynamin appear conserved in S2R+ cells. An Arf1- and Cdc42-dependent pathway in S2R+ cells that does not require Arf6, Rho or Rac1 As a basis for a molecular understanding of dynamin indepen- dent endocytosis in S2R+ cells, we first tested the involvement of Drosophila orthologues of several candidate genes known from other systems (Cdc42, Rho, Rac1, Arf6 and Arf1; [3]). Depletion of the corresponding transcript after dsRNA treatment was verified using rtPCR (Figure 6A), and cells were pulsed with Fdex and A568Tf and their normalized per cell uptake was quantitated as before. As expected from their roles in the secretory pathway, Arf1 and Cdc42 were required for the surface delivery of TfR (as determined by by the reduced surface Okt9 binding compared to control cells; Figure 6A). Strikingly, uptake of Tf when normalized to the lower surface levels of Tf receptor, was not affected in cells depleted of Arf1 or Cdc42. Instead, both genes were required for optimal levels of fluid phase uptake. Rac1, Rho or Arf6 depletion did not have major effects on uptake of either probe (Figure 6A). We examined Arf1-depleted cells at higher resolution because this gene has been shown to be directly involved in the early steps of endocytosis via GEECs in mammalian cells [10]. Consistent with an early role in the process, Arf1-depleted S2R+ cells contained fewer fluid endosomes after a short pulse, and the remaining endosomes were significantly reduced in intensity (Figure 6B). To test if the pathways of uptake of TfR and Fdex were indeed distinct as dynamin- and Arf1- dependent respectively, we co-depleted cells with dsRNA against both Arf1 and shibire. Fdex uptake was only reduced in the PLoS ONE | www.plosone.org 7 August 2009 | Volume 4 | Issue 8 | e6768