For fluorescence lifetime analysis, PAR-6 puncta were auto- matically detected and tracked as described [18]. The detection of puncta was set to be relatively sensitive, which results overall in fewer false negative and more frequent false positive detections. As a result, very short trajectories (#3 frames), which are more likely to represent individual or linked false positive detections, were excluded from analysis. Lifetime distribution/histogram and mean lifetime were extracted for n = 20 embryos for each condition. While the lifetime distributions of individual embryos are not normal distributions, the distribution of mean lifetimes measured in different embryos is normally distributed due to the Central Limit Theorem. Thus, the significance of differences of mean lifetimes between individual conditions could be determined using Student’s t-test. For the quantitation of F-actin cortical organization, regions of low fluorescence intensity were manually counted on 10 frames (each separated by 10 seconds) obtained from TIRF movies. Three embryos of each genotype were analyzed. Quantitation of the lifetimes of these low fluorescence intensity regions was done using the same method used to determine NMY-2 velocity, but tracking the regions of low dMoe::GFP fluorescence intensity instead. To measure spindle positioning, centrosome positions in DIC images were determined as described previously [13]. Supporting Information Figure S1 RAB-5 can be efficiently depleted by RNAi in early embryos. Midplane images of RAB-5::GFP in control(R- NAi), rab-5(RNAi) and rab-5 dyn-1(RNAi) embryos at pronuclear meeting, i.e., at the end of the phase of establishment of polarity. Quantitation of total cytoplasmic fluorescence intensity revealed a decrease in rab-5(RNAi) and in rab-5 dyn-1(RNAi) compared to control (p,0.001 in both cases, Student’s t-test; red stars). In all panels, anterior is to the left. Scale bars, 10 mm. (JPG) Figure S2 PAR-6 localization in rab-7(RNAi) and rab- 11(RNAi) embryos. Midplane images of PAR-6::GFP in control(RNAi), rab-7(RNAi) and rab-11(RNAi) embryos at pronuclear meeting, i.e., at the end of the phase of establishment of polarity. Quantitation of fluorescence intensity along the cortex reveals that the size of PAR-6::GFP cortical domain is similar in control and rab-7(RNAi) (p = 0.77, Student’s t-test) and enhanced in rab- 11(RNAi) (p = 4.97610205, Student’s t-test). In all panels, anterior is to the left. Scale bars, 10 mm. (JPG) Figure S3 Localization of endogenous PAR-6 and PKC-3 in rab-5(RNAi) embryos. Midplane images of control(RNAi) and rab-5(RNAi) embryos at pronuclear meeting, i.e., at the end of the polarity establishment phase, labelled with anti-PAR-6 or anti- PKC-3 antibodies. Quantitation of fluorescence intensity along the cortex revealed that the size of the PAR-6 cortical domain is reduced in rab-5(RNAi) embryos (p = 0.011, Student’s t-test; red star) compared to control(RNAi) embryos whereas the size of the PKC-3 cortical domain is similar in control(RNAi) and rab-5(RNAi) embryos (p = 0.41, Student’s t-test). In all panels, anterior is to the left. Scale bars, 10 mm. (TIF) Figure S4 The PAR-3/PAR-6/PKC-3 complex localizes to cytoplasmic puncta. (A) Midplane confocal images of fixed embryos expressing PAR-6::GFP and labelled with anti-PAR-3 and anti-PKC-3 antibodies during the phase of establishment of polarity. The white color indicates that all three markers co- RAB-5 Regulates PAR-6 Cortical Dynamics localize. Inset shows magnification of the boxed region. In all frames, anterior is to the left. Scale bar, 10 mm. (B) Midplane images of PAR-6::GFP and mCherry::RAB-7 in the cytoplasm of control embryos during the phase of establishment of polarity. The box is magnified 2.5-fold in inset. Fluorescence intensity was measured along the lines in each inset and represented for PAR-6 (green) or RAB-7 (red). No significant co-localization was observed (3.2% of co-localization, n = 53 puncta) compared to random control co-localization (3.1%, n = 31). (C) Midplane images of PAR-6::GFP and mCherry::RAB-11 in the cytoplasm of control embryos during the phase of establishment of polarity. The box is magnified 2-fold in inset. Fluorescence intensity was measured along the lines in each inset and represented for PAR-6 (green) or RAB-11 (red). No co-localization was observed (3.8% of co- localization with RAB-11 (n = 47 puncta) compared to random control co-localization (0%, n = 21). In all panels, anterior is to the left. Scale bars, 10 mm. (JPG) Movie S1 TIRF imaging of a wild-type embryo express- ing PAR-6::GFP during the polarity establishment phase. Images were captured at 1 second intervals and are played back at 10 images/second. (MOV) Movie S2 TIRF imaging of a wild-type embryo express- ing PAR-6::GFP during the polarity maintenance phase. Images were captured at 1 second intervals and are played back at 10 images/second. (MOV) Movie S3 TIRF imaging of a wild-type embryo express- ing PAR-6::GFP in which the fluorescence tracking software has been applied. Red spots correspond to fluorescence events. Images were captured at 1 second intervals and are played back at 10 images/second. Bar, 5 mm. (MOV) Movie S4 TIRF imaging of a rab-5(RNAi) embryo expressing PAR-6::GFP during the polarity establish- ment phase. Images were captured at 1 second intervals and are played back at 10 images/second. (MOV) Movie S5 TIRF imaging of a rab-5(RNAi) embryo expressing PAR-6::GFP during the polarity maintenance phase. Images were captured at 1 second intervals and are played back at 10 images/second. (MOV) Movie S6 TIRF imaging of a wild-type embryo express- ing dMoe::GFP during the polarity maintenance phase. Images were captured at 1 second intervals and are played back at 10 images/second. (MOV) Movie S7 TIRF imaging of a rab-5(RNAi) embryo expressing dMoe::GFP during the polarity maintenance phase. Images were captured at 1 second intervals and are played back at 10 images/second. (MOV) Strains used in this study. Table S1 (DOC) Acknowledgments We are grateful to Jeremy Nance, Ge´raldine Seydoux and the Caenorhabditis Genetics Center for strains and reagents. We also thank Paul Maddox, PLoS ONE | www.plosone.org 11 April 2012 | Volume 7 | Issue 4 | e35286