RAB-5 Regulates PAR-6 Cortical Dynamics Figure 4. PAR-6 co-localizes with endocytic markers at the cortex and in the cytoplasm. (A) Images of PAR-6::GFP and mCherry::RAB-5 at the cortex of control(RNAi) embryos during the polarity maintenance phase. The box is magnified three-fold in inset. Fluorescence intensity was measured along the line in each inset and represented for PAR-6 (green) or RAB-5 (red). The arrow points to a region with peaks of fluorescence intensity for each channel, and thus co-localization for both markers. (B) Images of PAR-6::mCherry and GFP::CHC-1 at the cortex of control(RNAi), rab- 5(RNAi), dyn-1(RNAi) and rab-5(RNAi); dyn-1(RNAi) embryos during the polarity maintenance phase. Graphical representations are as in panel A, except that PAR-6 is in red and CHC-1 is in green. The bar graph shows that co-localization of PAR-6::mCherry with GFP::CHC-1 is significantly increased in rab-5(RNAi) embryos compared to control(RNAi) (red star, p = 0.022, Student’s t-test) but not in dyn-1(RNAi) (p = 0.7) or in rab-5(RNAi); dyn-1(RNAi) (p = 0.89). The gray bars represent random co-localization control. n, number of puncta analyzed. Bars represent standard error of the mean. (C) Midplane images of PAR-6::GFP and mCherry::RAB-5 in the cytoplasm of control(RNAi) embryos during the polarity maintenance phase. Graphic representations are as in panel A. In all panels, anterior is to the left. Scale bars, 10 mm. doi:10.1371/journal.pone.0035286.g004 involvement of endocytic trafficking in this process. This is further supported by the report that turnover of cortical PAR-6 is slower in dyn-1(RNAi) embryos [8], which is compatible with our finding that PAR-6 cortical residence time is controlled by RAB-5 during embryonic polarization. However, the spindle positioning defect that we observed in rab-5(RNAi) embryos is distinct from that following depletion of DYN-1, and a recent report demonstrated that RAB-5 controls spindle positioning independently of DYN-1 [25]. We therefore determined whether other RAB-5-dependent phenotypes monitored in our analysis depend on DYN-1 function. We first tested whether the increase in PAR-6::mCherry and GFP::CHC-1 co-localization observed after depletion of RAB-5 is also observed after depleting DYN-1. We found that these two proteins co-localized to comparable levels in DYN-1-depleted embryos compared to control(RNAi) (Figure 4B; 29.164.8%, 197 puncta, 6 embryos for dyn-1(RNAi) compared to 26.663.7%, 528 puncta, 5 embryos for control(RNAi); p = 0.7), indicating that depleting DYN-1 alone does not modify their co-localization. However, the rab-5(RNAi)-dependent increase in PAR-6::mCherry and GFP::CHC-1 cortical co-localization was lost in embryos co- depleted for both RAB-5 and DYN-1 (Figure 4B; 27.463.6%, 251 puncta, 5 embryos; p = 0.89 when compared to control(RNAi) and p = 0,027 when compared to rab-5(RNAi)). This indicates that the increase in PAR-6::mCherry and GFP::CHC-1 co-localization observed after RAB-5 depletion requires the function of DYN-1. It PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e35286