GEEC Endocytosis in Drosophila Figure 8. Functional significance of GBF1 dynamics in the vicinity of the plasma membrane. (A) A CHO cell expressing low levels of GFPGBF1 imaged with widefield fluorescence microscopy (WF; left panel) or with TIRF microscopy (right panel) to reveal puncta close to the plasma membrane. (B) Fast sequential two-colour confocal imaging of a live CHO cell expressing GFPGBF1 and ABDRFP (activated Arf sensor; Kumari and Mayor, 2008). The colour merged image is a composite of the most peripheral plane of the cell in each channel (encompassing ,200 nm in the z-axis, 30 msec between each plane). Two boxed regions in the cell are magnified, the smaller as the merge inset, and the individual channels from the larger region are shown below. (C) Time lapse, sequential TIRF imaging of a CHO cell co-expressing GFPGBF1 and ABDRFP (left panels) or co-expressing GFPGBF1E794K and ABDRFP (right panels). The main reference images in each panel show dual-channel sets of TIRF images for a single time point, with a ,1 sec gap between channel acquisition. The insets in the reference images show the same plane imaged with widefield microscopy. The boxed regions are magnified and shown as a time series for each channel, with the time elapsed since the first reference frame displayed in seconds. Transient punctate or elongated structures that traverse the evanescent field and acquire both GFPGBF and ABDRFP over the time-lapse are circled in red. In the cell expressing GFPGBF1E794K, these structures persist for longer times in the evanescent field (magnified time series; right panel), but do not acquire ABDRFP. Quantitatively, cells expressing GFPGBF1E794K show significantly lower numbers of ABDRFP puncta in the evanescent plane (bar graph in right panel shows mean6s.e.m. values from 20 random planes from movies of 5 cells in each case, expressed as percentage of control).(D) CHO cells transfected with GFPGBF1E794K were treated with Brefeldin A (BFA) or carrier DMSO (control) and assayed for fluid phase (Rdex) uptake. Representative fields with GBF1E794K transfected (outlined in white) and untransfected cells, with or without BFA are shown in the left panel. The bar graph in the right panel shows the corresponding quantitation of fluid phase uptake as normalized to control, untransfected cells (mean6s.e.m.; n = 63,68,60,59; asterix denote P,0.05 by Student’s t-test). Bar in A,B = 10 mm, C = 2.5 mm, D = 20 mm. Insets in C are 0.25x, boxed regions are magnified 2x. doi:10.1371/journal.pone.0006768.g008 PLoS ONE | www.plosone.org 12 August 2009 | Volume 4 | Issue 8 | e6768