we found that PKC-3 localization is normal in RAB-5-depleted embryos, and therefore it is possible that PAR-2 is prevented from spreading toward the anterior cortex by PKC-3 activity despite PAR-6 being displaced toward the anterior. Such scenario would indicate that PKC-3 can function without PAR-6 at the cortex of RAB-5 depleted embryos. Additional experiments will be required to further address these issues. Finally, our results revealed that the cortical residence time of PAR-6 is short during both establishment and maintenance phases of embryonic polarity, and that depletion of RAB-5 results in a statistically significant increase in PAR-6 cortical residence time during the polarity maintenance phase. This suggests that a rapid turnover of cortical PAR-6 is required to accurately promote polarization. While it is currently unclear why a ,13% difference would have such effect on polarity, we note PAR-6 maximal fluorescence recovery after photobleaching was reported to be lower in embryos depleted for DYN-1 [8], consistent with this process being co-regulated by both proteins. Materials and Methods Strains and alleles All strains were maintained as described by Brenner [29]. They were grown at 15uC and assayed at 25uC after being grown at 25uC for at least 24 hours, unless otherwise stated. The wild-type strain was the N2 Bristol strain. The strains used in this study are presented Table S1. Double transgenic strains were generated by genetic crosses and each marker was followed either by phenotypic analysis or by PCR using specific primers. RNAi was performed on L4 animals using the feeding method as described [30], and worms were grown on RNAi plates for a minimum of 24 hours before collecting the embryos for phenotypic analysis. Except for RAB-5, whose depletion was directly determined by measuring fluorescence levels of a GFP fusion protein, the success of RNAi depletion for all proteins was based on assessing whether the resulting phenotype recapitulated published defects. Image acquisition For all imaging methods, embryos were obtained by cutting open gravid hermaphrodites using two 25-gauge needles and mounted individually using a mouth pipette on a coverslip coated with 0.1% poly-l-lysine in 10 ml of egg buffer [31]. The coverslip was placed on a 2% agarose pad and the edges were sealed with petroleum jelly. Detection of fluorescent proteins at the embryonic cortex was done using TIRF microscopy. Images were acquired by a Cascade II camera (Photometrics, Tucson, AZ, USA) mounted on a Nikon Eclipse Ti microscope (Nikon Canada Inc, Mississauga, ON, Canada). Samples were illuminated by 10 mW solid state lasers (488 nm and 561 nm, 10–30% power) and fluorescent light was collected by a Plan Apochromat 606/1.49 objective. Images were acquired at 1 second intervals for single channel imaging and at 3 seconds intervals for dual channel imaging and acquisition was controlled by Elements software (Nikon). Epifluorescence microscopy was used to measure the length of GFP::PAR-2, PAR-6::GFP or NMY-2::GFP cortical domains as well as the cytoplasmic co-localizations between PAR-6::GFP and either mCherry::RAB-5, mCherry::RAB-7 or mCherry::RAB-11. Images were acquired by a Zeiss HRM camera (Carl Zeiss Canada Ltd., Toronto, ON, Canada) mounted on a Zeiss Axio- Imager Z1 microscope. Samples were illuminated for 50–100 milliseconds by a mercury lamp and fluorescent light was collected by a Plan Apochromat 636/1.4 objective. Images were RAB-5 Regulates PAR-6 Cortical Dynamics acquired at 10 seconds intervals and the acquisition system was controlled by Axiovision software. Swept Field Confocal (SFC) microscopy was used to image cortical NMY-2::GFP and measure the velocity of cortical flows. Images were acquired by a CoolSNAP HQ2 camera (Photo- metrics) mounted on a Nikon SFC microscope (Nikon and Prairie Technologies, Madison, WI, USA) using the 45 mm pinhole setting. Samples were illuminated by 10 mW solid state lasers (488 nm and 561 nm, 10–30% power) and fluorescent light was collected by a 1006/1.4 Plan-Apochromat objective was used to acquire 16 confocal sections (separated by 0.5 mm) of the upper cortex exposed for 80 milliseconds at 10 second intervals. The acquisition system was controlled by Elements software (Nikon). Time-lapse Differential Interference Contrast (DIC) microscopy was done using Plan Apochromat 636/1.4 or 1006/1.4 objectives mounted on a Zeiss Axioimager Z1 microscope, as described above. Images were acquired at 10 seconds intervals. Image analysis was performed using ImageJ software (http:// rsbweb.nih.gov/ij/). Positioning of the first mitotic spindle and asynchrony between AB and P1 cells were quantitated as previously described [13]. Image analysis The co-localization between fluorescently-tagged PAR-6 and other fluorescent markers (CHC-1, RAB-5, RAB-7, RAB-11, dMoe) was determined using ImageJ software (NIH). A 10 pixel- thick line crossing a cortical or cytoplasmic PAR-6 punctum was drawn and mean fluorescence intensity profiles along this line were obtained and compared for both fluorescent channels. Proteins were deemed at co-localizing when a peak of PAR-6 fluorescence intensity was directly superimposed with that of the other fluorescent marker. Quantitation of random co-localization control was done similarly, except that the images were rotated along the horizontal axis for one of the two markers. To quantitate the size of PAR-2, PAR-6, PKC-3 or NMY-2 cortical domains, ImageJ software was used to draw a 10 pixel- thick line on the whole perimeter of the embryos and plot the corresponding mean fluorescence intensity profiles. The mean fluorescence intensity values were interpolated as the percentage of total perimeter for each embryo. In all cases 0% = anterior pole and 100% = posterior pole. Intensities were represented as the percentage of maximum intensity along the perimeter. The length of domains were calculated as the length of embryo perimeter with intensity values superior to a certain threshold (30% of maximal intensity for PAR-6::GFP and NMY-2::GFP, 40% for PAR-6 and PKC-3 or 60% of maximal intensity for PAR-2::GFP). Depletion of RAB-5 by RNAi induced a discontinuity in the NMY-2::GFP domain, leading to the appearance of small cortical patches where fluorescence intensity was below the threshold within a region of high fluorescence intensity. These patches of low fluorescence intensity that were bordered by regions of higher fluorescence intensity were considered as superior to the threshold to properly determine domain length. In these embryos, fluorescence intensity below the threshold was therefore only considered significant when it was measured for a continuous region larger than 2 mm. To quantitate the velocity of NMY-2 foci during polarization, we generated kymographs using ImageJ software by drawing a line from the anterior to the posterior pole of the embryos. Three kymographs were created for each embryo (one on each side and one in the center of the embryo) and the 12 most posterior foci present in these kymographs were used for quantitation. Velocity was calculated by reporting the distance traveled by these foci over time. PLoS ONE | www.plosone.org 10 April 2012 | Volume 7 | Issue 4 | e35286