RAB-5 Regulates PAR-6 Cortical Dynamics with peaks of fluorescence intensity for each channel, and thus co-localization for both markers. The bar graph shows that co-localization of PAR- 6::mCherry with dMoe::GFP is significantly increased in rab-5(RNAi) embryos compared to control(RNAi) (red star, p = 0.013, Student’s t-test). The gray bars represent random control co-localization. n, number of puncta analyzed. Bars represent standard error of the mean. White arrows point to regions of LFI that are visible with both markers and can be superimposed. (D) Model depicting the regulation of PAR-6 function by RAB-5 (See discussion for details). doi:10.1371/journal.pone.0035286.g005 further demonstrates DYN-1 levels were duly depleted in rab- 5(RNAi); dyn-1(RNAi) embryos. We then determined whether depleting DYN-1 affected the general organization of the actin cytoskeleton, as is the case for RAB-5 depletion. We found that upon DYN-1 depletion, the number of regions with low dMoe::GFP fluorescence intensity was comparable to control(RNAi) embryos (55611 regions; p = 0.98) and significantly lower than in rab-5(RNAi) embryos (p = 0.008; Figure 5A). Interestingly, these regions of dMoe::GFP fluorescence intensity were also observed in abnormally high numbers (134612 regions) in embryos co-depleted for both DYN-1 and RAB-5, indicating that RAB-5 regulates the organization of the actin cytoskeleton independently of DYN-1. While it remains formally possible that, under our RNAi conditions, DYN-1 is sufficiently depleted to reveal its role in regulating the association between PAR-6 and CHC-1 but that further depletion would be required to observe effects on the actin cytoskeleton, we note that the polarity defects associated with DYN-1-depletion are observed under our RNAi conditions and therefore we consider this as unlikely. Together, these results support a model in which RAB-5 regulates the organization of the actin cytoskeleton independently of DYN-1 function. Discussion Our findings reveal that RAB-5 plays a significant role in polarization of the early C. elegans embryo. We showed that depleting the endocytic regulator RAB-5 perturbs the size of PAR- 6 domain during the polarity establishment phase, as well as PAR- 6 dynamics and cortical organization during the phase of maintenance of polarity. Depletion of RAB-5 suppressed the polarity phenotypes of par-2 mutants, indicating that its activity becomes critical when polarity is compromised and that its role in this process is biologically relevant. Furthermore, RAB-5 depletion disrupted the organization of cortical actin and altered the co- localization of PAR-6 with components such as clathrin heavy chain at the embryonic cortex. Based on these results, we propose that RAB-5 controls PAR-6 localization and cortical dynamics by regulating the organization of the actin cytoskeleton and the association of PAR-6 (and perhaps other anterior PAR proteins) with cortical components. Depleting RAB-5 did not perturb cortical NMY-2 flows at the posterior cortex during the establishment phase of polarity. On the other hand, it had a significant effect on the duration of PAR-6 residence time at the cortex during the polarity maintenance phase. Together, these results suggest that RAB-5 exerts its activity mainly during polarity maintenance. However, we note that PAR-6 localization was affected during the polarity establishment phase in rab-5(RNAi) embryos, indicating that RAB-5 could also act during this time. As anterior PAR proteins are themselves positive regulators of endocytosis [26], our results reflect the possible existence of a feedback mechanism between RAB-5 and PAR proteins to regulate polarity and actin organization. Previous work showed that depletion of dynamin results in a small spindle-positioning defect in the 1-cell embryo and in a decrease in PAR-6 dynamics at the cortex [8]. Together with our quantitative analysis of PAR-6 association with early endosomal markers, this supports the notion that part of RAB-5’s role in cell polarity occurs through its function in endocytosis and early endosome formation. However, several lines of evidence suggest that RAB-5 has a broader role in the regulation of cell polarity, as depleting dynamin did not recapitulate all of the defects observed following RAB-5 depletion. For instance, the posterior over- displacement phenotype of the spindle that we observed in RAB-5- depleted embryos is opposite from the anterior positioning defect reported for dynamin-depleted embryos [8,25]. Furthermore, we found that the defects in PAR-6 and actin cortical organization observed after RAB-5 depletion were absent from embryos depleted in dynamin but were still visible in embryos co-depleted for RAB-5 and DYN-1, indicating that RAB-5 controls cortical organization independently of DYN-1 function. We therefore propose a model (Figure 5D) in which RAB-5, along with dynamin, controls polarity through its function as an endocytic regulator by regulating the cortical trafficking of PAR-6, but has other, dynamin-independent functions in ensuring correct spindle positioning and in maintaining proper actin and PAR-6 cortical organization. While it is currently unclear how this additional, dynamin-independent function of RAB-5 in cell polarity is related to its role in endocytosis, it is compatible with the idea of early endosomes acting as hubs for intracellular signaling, as has been recently suggested [27]. Our results revealed that PAR-6 co-localizes robustly with clathrin heavy chain at the cortex of rab-5(RNAi) embryos. While we also observed co-localization between these two proteins in wild-type embryos, the value was not significantly different from random co-localization control measurements (p = 0.09). This suggests that the association of PAR-6 with the early endocytic machinery is transient and highly dynamic in wild-type embryos, and that depletion of RAB-5 increases the time of association in a dynamin-dependent manner. This is compatible with the role of RAB-5 in regulating early endosome formation and endocytic trafficking. Alternatively, it is possible that depleting RAB-5 confers novel properties to cortical components that would promote such association. Our results revealed that RAB-5 controls the anterior localization of PAR-6 but has no effect on the localization of PAR-2. This is surprising given that anterior and posterior PAR proteins have been shown to localize to mutually exclusive cortical domains. Why does PAR-2 posterior localization remain un- changed despite the over-anteriorization of PAR-6? We found that cortical flows are normal in RAB-5-depleted embryos, suggesting that polarization occurs normally and that most defects are observed during the maintenance phase of polarity. This leads us to speculate that RAB-5 would not be required for the localization of PAR-2 during the polarization phase, but would be necessary for its trafficking dynamics during maintenance phase, such that its localization to cortices that are free of PAR-6 would be more difficult without RAB-5 function, and perhaps endocytosis function. The possibility that RAB-5 is involved in the trafficking of PAR-2 is further supported by our observation that the transient anterior PAR-2 crescent is lost in rab-5(RNAi) embryos. Such anterior accumulations of PAR-2 were previously reported to depend on the meiotic spindles and the mid-body remnants following meiotic cytokineses [28], and our results suggest that their presence could require dynamic endocytosis. Alternatively, PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e35286