GEEC Endocytosis in Drosophila Figure 4. A dynamin insensitive endocytic machinery operates in S2R+ cells. (A) Specific depletion of Drosophila dynamin by dsRNA against the shibire (shi) gene as shown by Western detection of cell homogenates after 4 days depletion (left panel). Cells were also processed for immunocytochemistry followed by TIRF imaging of dynamin puncta close to the plasma membrane (right panel). (B) Cell populations were depleted of dynamin or control (zeocin) dsRNA for 4 days, and uptake of Fdex, Tf as well as surface TfR levels (Okt) were quantitated. Representative cells (outlined) from each dsRNA population are shown in all three channels and the bar graph (right panel) shows population means6s.e.m. of 2 independent experiments with .200 cells in each condition. A quantitation of internalized GFP-GPI under the same conditions is shown alongside. (C) A quantitative surface accessibility assay [19] was employed to examine scavenger receptor availability in shibire (shi) depleted S2R+ cells. The assay measures the extent to which receptors labeled with biotinylated Cy3mBSA (Cy3bmBSA) are accessible to exogenous Cy5-Streptavidin (Cy5SA; see cartoon) after they are allowed to internalize the labeled ligand (x axis legends: Cy3 - binding capacity; Cy5SA - surface accessibility to Cy5-SA; and Cy5SA/Cy3 - relative surface accessibility). The bar graph shows that after 3 min of pulse followed by 10 min of chase, shi depleted cells show ,twofold higher levels of Cy5SA binding when compared to controls (mean6s.e.m.; 2 experiments, .30 cells in all cases), although they bind the same amount of Cy3bmBSA. Furthermore, while Fdex continued to be internalized and trafficked towards the center of shi-depleted cells (filled arrowheads in right panel), the large majority of Cy3bmBSA labeled structures were found at the cell periphery, largely accessible to Cy5SA (empty arrowheads). By this time, in control cells, Cy3bmBSA and Fdex had mixed extensively in central structures which were inaccessible to Cy5SA (marked with filled arrowheads). (D) The bar graph shows normalized uptake of Fdex, TfR and GFP-GPI in S2R+ cells treated with 20 mM dynasore (dyn) for 20 min or carrier DMSO alone (mock). Values are mean6s.e.m. (.50 cells in all cases) from two independent experiments. Representative images in the right panel are scaled equally to show the marked difference in Okt9 levels in cells after dynasore treatment, while Fdex intensities are unchanged or higher compared to the mock treatment. Bars in A = 2.5 mm, B = 20 mm, C = 5 mm, D = 20 mm. doi:10.1371/journal.pone.0006768.g004 PLoS ONE | www.plosone.org 8 August 2009 | Volume 4 | Issue 8 | e6768