RAB-5 Regulates PAR-6 Cortical Dynamics Figure 3. RAB-5 controls PAR-6 residence time at the cortex. (A) Time lapse images of PAR-6::GFP in the middle plane and cortical plane of control(RNAi) embryos. Imaging at the cortical plane was done by TIRF microscopy. A schematic representation of PAR protein localization in each plane is depicted on the left. (B) Magnified images of PAR-6::GFP at the cortex of control(RNAi) embryos obtained by TIRF microscopy. The white arrow points to a PAR-6-positive puncta appearing and disappearing from the focal plane during this 9-second excerpt. (C) TIRF images of cortical PAR-6::GFP in a control(RNAi) embryo before (upper image) and after (lower image) processing by particle tracking software. Each red dot on the bottom image is recognized as a PAR-6-positive structure. Quantitative automated analysis of PAR-6::GFP puncta shows that they have short cortical residence time during both the establishment and maintenance phases of polarity. The mean cortical residence time is significantly longer during the establishment phase than during the maintenance phase (red star, p = 0.003, Student’s t-test). Error bars represent standard deviation. (D) TIRF images of cortical PAR-6::GFP in rab-5(RNAi) embryos. Quantitative automated analysis of PAR-6::GFP puncta shows that depletion of RAB-5 results in a significant increase in their mean cortical residence time during the maintenance phase (red star, p = 0.026, Student’s t-test), but not during the establishment phase of polarity. Error bars represent standard deviation. White arrows point to cortical regions where PAR-6::GFP is excluded. In all panels anterior is to the left. Scale bars, 10 mm. doi:10.1371/journal.pone.0035286.g003 PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e35286