PICK1 associates transiently with the Golgi compartment and is capable of tubular deformation of liposomes in vitro.

Abstract

<p>(A) Brief (5 min) brefeldin A treatment traps PICK1 in the trans-Golgi. Confocal images of GH1 cells immunostained for PICK1, and the trans-Golgi marker TGN38, and the trans-Golgi/immature vesicle marker syntaxin 6 before (top) and after 5 min BFA treatment (bottom). Panels show from left signal from immunolabeled PICK1 (Alexa Fluor 568 signal), signal from TGN38 (Alexa Fluor 488 signal), signal from syntaxin 6 (Alexa Fluor 647 signal), and overlay of the three channels. Insets highlight an area with overlapping localization of PICK1 and syntaxin 6 but with PICK1 adjacent to TGN38 (top) and colocalization of PICK1 with both syntaxin and TGN38 (bottom). (B) Quantification of the PICK1 colocalization with TGN38 and syntaxin 6 after 5 min BFA treatment using Van Steensel's cross-correlation function, which reports the Pearson cross-correlation as a function of the relative movement of the two channels with respect to each other. Both syntaxin 6 and TGN38 show a sharp peak of similar height close to Δx = 0, indicating specific co-localization. Co-localization was quantified for 10–20 cells from three independent experiments, and data are means ± SE. Note that the drop in peak values compared to no BFA (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001542#pbio-1001542-g006" target="_blank">Figure 6F</a>) likely reflects the increased diffuse localization of both markers after BFA treatment. (C) Fast time-lapse dual color live confocal imaging showing transient association of YFP-PICK1 BAR with the Golgi marker GalT-Cerulean. Images are maximal intensity projections of representative time-lapse series for YFP-PICK1 BAR (left), YFP PICK1 BAR V121E-L125E (middle), and YFP-PICK1 BAR 3KE (right). The gray scale projections (left images) show the YFP channel alone, indicating highly dynamic behavior of YFP PICK1 BAR (left) and more static behaviour of YFP-PICK1 BAR V121E-L125E (middle) punctae, whereas no punctae are seen for YFP-PICK1 BAR 3KE (right). The dual color projections (right images, YFP channel in yellow, Cerulean channel in blue) show that most of the activity for YFP-PICK BAR (left) is lining the Golgi, whereas YFP-PICK1 BAR V121E-L125E (middle) shows activity throughout the cell. Circles indicate transiently appearing punctate structures (lasting for less than 10 frames, ∼30 s). Crosses indicate stable punctate structures lasting for more than 10 frames. (D) Time-lapse series of two transient punctate structures of YFP-PICK1 BAR, and (E) two stable punctate structures of YFP-PICK1 BAR V121E-L125E. Scale bar, large images 5 µm, time lapse 1 µm. (F) Tubulation of surface-immobilized artificial giant membrane vesicles by the PICK1 BAR domain. Purified and Alexa Fluor 488–labeled GST-PICK1 BAR or GST-PICK1 BAR V121E-L125E (control) with impaired membrane binding capacity. Protein was incubated at room temperature with surface immobilized and fluorescently labeled vesicles before confocal fluorescent imaging. No effect was seen for the mutant, whereas massive tubulation was seen with GST-PICK1 BAR. Arrow indicates a budding vesicle from a single GV.</p