RAB-5 Regulates PAR-6 Cortical Dynamics Figure 2. RAB-5 controls PAR-6 cortical localization independently of actomyosin contractility. (A) Midplane images of PAR-6::GFP in control(RNAi) and rab-5(RNAi) embryos at pronuclear meeting, i.e., at the end of the establishment phase of polarity. Quantitation of fluorescence intensity along the circumference of the cortex reveals that the size of PAR-6::GFP cortical domain is more anterior in rab-5(RNAi) embryos compared to control(RNAi) (red star, p = 6.11610206, Student’s t-test). (B) Midplane images of GFP::PAR-2 in control(RNAi) and rab-5(RNAi) embryos at pronuclear meeting. Quantitation of fluorescence intensity along the cortex shows that the size of GFP::PAR-2 cortical domain is similar in control(RNAi) and rab- 5(RNAi) embryos (p = 0.98, Student’s t-test). Quantitation excluded the anterior crescent of GFP::PAR-2 visible in wild-type embryos (arrow) but absent from rab-5(RNAi) embryos. (C) Images of NMY-2::GFP at the mid-plane of control(RNAi) and rab-5(RNAi) embryos at pronuclear meeting. Quantitation of fluorescence intensity along the cortex shows that the size of NMY-2::GFP cortical domain is similar in control(RNAi) and rab-5(RNAi) embryos (p = 0.17, Student’s t-test). (D) Images of NMY-2::GFP at the cortex of control(RNAi) and rab-5(RNAi) embryos during the phase of establishment of polarity. Kymographs were generated along the antero-posterior axis (orange lines). The average velocity of NMY-2 puncta is similar in control(RNAi) and rab-5(RNAi) embryos (p = 0.86, Student’s t-test). In each frame anterior is to the left and white arrowheads indicate the boundary of cortical fluorescence. n, number of embryos analyzed. Error bars represent standard deviation. Scale bars, 10 mm. doi:10.1371/journal.pone.0035286.g002 planes [4,15] (Figure 3A). As shown in Figure 3A and 3B, TIRF imaging revealed that PAR-6::GFP is visible in early embryos in individual puncta of various sizes and intensities that appear and disappear from the focal plane in a highly dynamic manner (Figure 3B and Movies S1, S2). We quantitated PAR-6 cortical dynamics using an automated computational approach based on tracking individual PAR-6::GFP puncta (Figure 3C and Movie S3). This approach allowed us to simultaneously track hundreds of puncta per embryo. We found that PAR-6::GFP puncta displayed on average very short residence times at the cortex. The cortical residence time of PAR-6::GFP was significantly longer during the establishment phase of polarity (9.261.0 sec) than during the maintenance phase (8.360.9 sec; p = 0.003), consistent with the hypothesis that these two phases are governed by distinct mechanisms (Figure 3C). Interestingly, depleting RAB-5 perturbed the dynamics of PAR-6::GFP puncta during the maintenance phase of polarity and resulted in an increase in their residence times at the cortex (and perhaps also their overall size) when compared to control(RNAi) embryos (9.461.8 sec; p = 0.025) (Figure 3D and Movies S4, S5). RAB-5 depletion had no statistically significant effect on cortical PAR-6::GFP dynamics during the establishment phase of polarity (10.061.7 sec; p = 0.112), suggesting that RAB-5 activity contributes to polarity mainly during the maintenance phase. Therefore, we conclude that PAR-6 is a highly dynamic cortical protein whose cortical PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e35286