has demonstrated that the GTPase dynamin, an upstream regulator of endocytosis, affects the polarization of the C. elegans embryo [8], the contribution of other endocytic regulators in this process is unclear. Here, we studied the function of endocytic regulators in polarization of the C. elegans embryo. We find that depleting RAB-5, a small GTPase that functions as the main regulator of early endosome formation, perturbs the organization and function of PAR-6 in C. elegans during the maintenance phase of polarity. Interestingly, while depletion of RAB-5 recapitulated some of the phenotypes observed in embryos depleted for dynamin, it also resulted in unique spindle positioning and cortical organization defects. Our results support a model in which RAB-5 controls C. elegans early embryonic polarity in both dynamin- dependent and -independent manners, by regulating the cortical localization of PAR-6 and the organization of the actin cytoskeleton. Results Depletion of RAB-5 and other endocytic regulators results in early embryonic polarity phenotypes We hypothesized that endocytic regulators could contribute to early C. elegans embryonic polarization by regulating the cortical localization and/or function of anterior PAR proteins. To characterize this involvement, we focused our analysis on three small GTPases, known to be essential regulators of endocytic trafficking, RAB-5, RAB-7 and RAB-11, which respectively function as master regulators of early, late and recycling endosome function [9]. We first depleted each of these three genes by RNAi and monitored the first two divisions of C. elegans embryos by time- lapse differential interference contrast (DIC) microscopy, quanti- tating two polarity related phenotypes: positioning of the first mitotic spindle, which is typically displaced toward the posterior pole of the cell, and division asynchrony of the AB and P1 blastomeres at the two-cell stage, as AB typically divides 120 seconds before P1 in wild-type embryos. We found that depletion of RAB-5 or RAB-7 in a wild-type background affected both phenotypes and resulted in the first mitotic spindle forming more toward the posterior pole and in an increase in the asynchrony between AB and P1 division as compared to wild-type embryos (Figure 1). As previously reported [10], depletion of RAB- 11 also affected both phenotypes, although the penetrance was more variable, with some embryos having their first spindle overly shifted to the posterior, as for RAB-5-depleted embryos, and other embryos showing defects consistent with a loss of polarity. These results indicate that depletion of RAB-5, RAB-7 and RAB-11 regulate polarity-dependent processes in the early C. elegans embryo. Previous studies showed that regulators of C. elegans polarity can be revealed by functionally assessing whether their depletion can modify the embryonic phenotype of par-2(it5ts) mutants [11,12,13,14]. For instance, depleting genes that positively regulate the amount of PAR-6 protein can result in suppression par-2 mutant phenotypes, while negative regulators enhance the defects of par-2 mutants. We therefore scored spindle positioning and cell division timing after depletion of each of the three endocytic regulators in par-2(it5ts) mutant embryos, in which the first mitotic spindle is central and, at the two-cell stage, the blastomeres divide synchronously (Figure 1). We found that depletion of RAB-5 efficiently suppressed these polarity defects in par-2(it5ts) mutant embryos grown at restrictive temperature (Figure 1), making it one of the strongest suppressors of par-2 defects identified to date [14]. Depletion of either RAB-7 or RAB- 11 in par-2(it5ts) mutants modified the cell division synchrony RAB-5 Regulates PAR-6 Cortical Dynamics phenotype of par-2(it5ts) mutants but had no influence on the spindle positioning defect (Figure 1). Together, these results indicate that while several endocytic regulators contribute to polarization of the C. elegans embryo, RAB-5 could play a functionally important role in this process and we therefore focused our analysis on the role of this protein in embryonic polarization. RAB-5 controls PAR-6 cortical localization independently of actomyosin contractility As polarization of the early embryo depends on the asymmetric localization of PAR proteins, we tested whether depletion of RAB- 5 by RNAi (Figure S1) perturbed the localization of anterior and posterior PAR protein cortical domains. We measured the distribution of PAR-6::GFP or GFP::PAR-2 at the time of pronuclear meeting, i.e., at the onset of the polarity maintenance phase, by quantitating the fluorescence intensity of each GFP tagged protein along the cortex (Figure 2). We found that PAR- 6::GFP occupies 59% of the anterior cortex of wild-type embryos, as previously described [13,15]. Interestingly, PAR-6::GFP localized to a smaller domain in embryos depleted of RAB-5 and covered 50% of the anterior cortex (Figure 2A), a phenotype that was not observed in embryos depleted for RAB-7 or RAB-11 (Figure S2). While anterior enrichment of endogenous PAR-6 was also observed by immunofluorescence on fixed rab-5(RNAi) specimen, no difference was observed for the localization of endogenous PKC-3 between control(RNAi) and rab-5(RNAi) em- bryos (Figure S3), and, the length of the PAR-2::GFP domain was also similar in control(RNAi) and rab-5(RNAi) embryos (Figure 2B). These results suggest that RAB-5 affects the localization of PAR-6 independently of other PAR proteins (see discussion). In addition to its posterior localization, PAR-2 is transiently present at the anterior cortex near the site of meiotic cytokinesis in 42% of wild- type embryos (n = 12), as previously reported [16,17]. This anterior PAR-2 cap was observed in only 11% of rab-5(RNAi) embryos (n = 18). These results indicate that RAB-5 acts as a positive regulator of PAR-6 to regulate its activity and localization along the antero-posterior axis. Localization of PAR-6 to the anterior cortex relies on cortical flows driven from the posterior cortex to the anterior cortex by non-muscle myosin II (NMY-2) activity [4]. Therefore, it is possible that depletion of RAB-5 results in an over-anteriorized PAR-6 domain by affecting cortical flows. To test this possibility, we measured the distribution of NMY-2::GFP along the antero- posterior axis of the embryonic cortex at pronuclear meeting. In control(RNAi) embryos, NMY-2 is enriched at the anterior of the embryo at the end of the polarization phase [4] and we found that this phenotype was not affected by depletion of RAB-5 (Figure 2C). We also quantitated the velocity of cortical NMY-2::GFP movement toward the anterior pole in time-lapse acquisitions and found no difference between control(RNAi) and rab-5(RNAi) embryos, in both of which myosin moved at an average speed of 4.5 mm/min (Figure 2D). Together, these results indicate that RAB-5 controls the anterior localization of PAR-6 independently of cortical NMY-2 flows during the polarity establishment phase. RAB-5 regulates PAR-6 residence time at the cortex In C. elegans and other systems, RAB-5 was shown to be a critical regulator of endocytosis by controlling the formation and function of early endosomes. We therefore hypothesized that RAB-5 promotes PAR-6 localization through its role in the regulation of early endosome function. To test this, we devised a new method, based on total internal reflection fluorescence (TIRF) microscopy, to monitor the trafficking of PAR-6::GFP at the cell cortex. TIRF PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e35286