residence time is controlled by RAB-5 during embryonic polarization. PAR-6 partially co-localizes with endocytic markers at the cortex and in the cytoplasm Among the proteins present at the cortex, components of the early endocytic pathway have been shown to be extremely dynamic with high variance in residence times. In mammals for instance, such components are typically visible by TIRF microscopy for periods ranging from 5 to 100 seconds [18]. Furthermore, in C. elegans, some endocytic regulators are known to be enriched at the anterior of the embryo where anterior PAR proteins localize [19]. Therefore the high, RAB-5-dependent cortical dynamics of PAR-6 could reflect an important association of the PAR proteins at the cortex with endocytic organelles. To address this possibility, we generated double transgenic strains co- expressing fluorescently-tagged PAR-6 (GFP or mCherry) with RAB-5 fused to mCherry (mCherry::RAB-5) or clathrin heavy chain fused to GFP (GFP::CHC-1), and used TIRF microscopy to analyze their co-localization at the cortex during the polarity maintenance phase. One caveat is that our analyses may be based on protein overexpression, although this is typically mild in the C. elegans germline and embryo. Quantitation of fluorescence intensities revealed that 18.861.4% of PAR-6::GFP puncta co- localized with mCherry::RAB-5 at the cortex (Figure 4A; 182 puncta, 4 embryos). This was significantly higher than the control (6.361.4%; 64 puncta, 4 embryos; p = 0.0002) in which random co-localization was determined using the same analysis performed with one of the two images flipped horizontally. By contrast, we observed that 26.663.7% of PAR-6::mCherry puncta co-localized with GFP::CHC-1 positive structures (Figure 4B; 528 puncta, 5 embryos), a value that is higher but not statistically significantly different than the random co-localization control (17.962.6%; 146 puncta, 5 embryos, p = 0.09). Interestingly, depletion of RAB-5 resulted in a significant increase in PAR-6::mCherry and GFP::CHC-1 co-localization compared to control(RNAi) (Figure 4B; 38.161.7%, 651 puncta, 5 embryos; p = 0.02). This was significantly higher than random co-localization control (14.663.9%; 79 puncta, 4 embryos, p = 0.0006), indicating that the increase in co-localization is specific for RAB-5 depletion. These results indicate that RAB-5 regulates the association of PAR-6 with early endocytic regulators at the cortex. While PAR-6::GFP mainly localizes to the cortex in early embryos, we observed that low amounts are also visible as cytoplasmic puncta during the polarization phase. Interestingly, we found that 37.9% of PAR-6::GFP cytoplasmic puncta co- localized with mCherry::RAB-5 (Figure 4C; 49 puncta, 11 embryos), a value significantly higher than random co-localization control (9.364.7%; 49 puncta, 3 embryos). PAR-6 also partially co-localized in the cytoplasm with EEA-1, another marker of early endosomes (data not shown). Furthermore, immunofluorescence experiments on fixed specimens revealed that some of these PAR- 6::GFP cytoplasmic structures can also contain PAR-3 and PKC- 3, suggesting that all anterior PAR proteins can be found in endosomal compartments (Figure S4). We did not observe any co- localization of PAR-6::GFP with either mCherry::RAB-7 or mCherry::RAB-11, indicating specificity of PAR-6 association with the early endosome compartment (Figure S4). These results indicate that PAR-6 and possibly other anterior PAR proteins associate with early endosomes during embryonic polarization and that such association is likely to be important for the regulation of polarity. RAB-5 Regulates PAR-6 Cortical Dynamics RAB-5 controls actin and PAR-6 organization at the cortex In addition to its role in early endocytosis and in the function of early endosomes, RAB-5 has been shown to control actin remodeling in mammalian cells [20,21]. Depletion of C. elegans RAB-5 was also reported to mildly affect the actomyosin cytoskeleton in addition to severely perturbing the morphology of the endoplasmic reticulum [22]. As the actin cytoskeleton typically mediates the cortical organization of PAR proteins, we hypothesized that RAB-5 may control C. elegans embryonic polarity through its role in actin remodeling. Indeed, we observed that PAR-6::GFP cortical organization is perturbed in rab-5(RNAi) embryos, in which it appears to be excluded from sub-regions of the anterior cortex during the maintenance phase of polarity (Figure 3D, arrows). We therefore analyzed the effect of RAB-5 depletion on actin organization. TIRF imaging of embryos expressing the actin-binding domain of dMoesin fused to GFP (dMoe::GFP; [23]) revealed that RAB-5 depletion results in disorganization of the actin cytoskeleton. Specifically, dMoe::GFP qualitatively appeared less filamentous and more punctate than in control(RNAi) embryos. After depletion of RAB-5, we also observed the appearance of several regions of lower dMoe::GFP fluores- cence intensity (Figure 5A). These regions were observed more frequently in rab-5(RNAi) embryos (16164 regions per 10 frames) than in control(RNAi) (5568 regions per 10 frames; p = 0.0003). Kymograph analysis revealed that these regions are dynamic in both conditions but that their dynamics are slower in rab-5(RNAi) embryos compared to control(RNAi) (Figure 5B and Movies S6, S7). Such disorganization was mainly observed during the mainte- nance phase of polarity, after completion of the contractility-based polarity establishment phase. A similar cortical disorganization was also observed at the end of the polarity establishment phase in RAB-5-depleted embryos expressing NMY-2::GFP (Figure 2C). This demonstrates that rab-5 depletion leads to a disorganization of cortical PAR-6 and, more generally, of the actin cytoskeleton. To determine the links between the organization of PAR-6 and the actin cytoskeleton, we generated a strain co-expressing PAR- 6::mCherry and dMoe::GFP and monitored the localization of both cortical markers during the polarity maintenance phase using TIRF microscopy. We found that 38.7%64.1 of the PAR- 6::mCherry puncta co-localized with dMoe::GFP (Figure 5C; 636 puncta, 6 embryos) and this was significantly higher than the random control (23.8%62.3; 143 puncta, 6 embryos; p = 0.01). Co-localization of PAR-6 and dMoe was significantly increased in rab-5(RNAi) embryos when compared to control(RNAi) (Figure 5C; 54.666.2%, 517 puncta, 5 embryos; p = 0.01), whereas no change was found when measuring co-localization in the random control samples (19.9%62.8; 119 puncta, 5 embryos; p = 0.29). This indicates that depletion of RAB-5 increases the co-localization between PAR-6 and F-actin. Furthermore, we observed that the regions of low dMoe::GFP fluorescence intensity were also largely devoid of PAR-6::mCherry fluorescence signal (Figure 5C, arrows). This indicates that RAB-5 could regulate PAR-6 organization and dynamics by controlling its association with F- actin and the general organization of the actin cytoskeleton. RAB-5 has DYN-1-dependent and –independent functions in the early embryo Our results support a role for RAB-5 in the regulation of PAR-6 cortical dynamics during C. elegans embryonic polarization. DYN- 1, the C. elegans ortholog of dynamin, has been reported to exist in a complex with RAB-5 [24], and was previously implicated in regulation of embryonic polarity [8], suggesting a broader PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e35286