RAB-5 Controls the Cortical Organization and Dynamics of PAR Proteins to Maintain C. elegans Early Embryonic Polarity

In all organisms, cell polarity is fundamental for most aspects of cell physiology. In many species and cell types, it is controlled by the evolutionarily conserved PAR-3, PAR-6 and aPKC proteins, which are asymmetrically localized at the cell cortex where they define specific domains. While PAR proteins define the antero-posterior axis of the early C. elegans embryo, the mechanism controlling their asymmetric localization is not fully understood. Here we studied the role of endocytic regulators in embryonic polarization and asymmetric division. We found that depleting the early endosome regulator RAB-5 results in polarity-related phenotypes in the early embryo. Using Total Internal Reflection Fluorescence (TIRF) microscopy, we observed that PAR-6 is localized at the cell cortex in highly dynamic puncta and depleting RAB-5 decreased PAR-6 cortical dynamics during the polarity maintenance phase. Depletion of RAB-5 also increased PAR-6 association with clathrin heavy chain (CHC-1) and this increase depended on the presence of the GTPase dynamin, an upstream regulator of endocytosis. Interestingly, further analysis indicated that loss of RAB-5 leads to a disorganization of the actin cytoskeleton and that this occurs independently of dynamin activity. Our results indicate that RAB-5 promotes C. elegans embryonic polarity in both dynamin-dependent and -independent manners, by controlling PAR-6 localization and cortical dynamics through the regulation of its association with the cell cortex and the organization of the actin cytoskeleton

DHTP is an allosteric inhibitor of the kinesin-13 family of microtubule depolymerases

AbstractThe kinesin-13 family of microtubule depolymerases is a major regulator of microtubule dynamics. RNA interference-induced knockdown studies have highlighted their importance in many cell division processes including spindle assembly and chromosome segregation. Since microtubule turnovers and most mitotic events are relatively rapid (in minutes or seconds), developing tools that offer faster control over protein functions is therefore essential to more effectively interrogate kinesin-13 activities in living cells. Here, we report the identification and characterization of a selective allosteric kinesin-13 inhibitor, DHTP. Using high resolution microscopy, we show that DHTP is cell permeable and can modulate microtubule dynamics in cells

PAR-6 co-localizes with endocytic markers at the cortex and in the cytoplasm.

<p>(<b>A</b>) Images of PAR-6::GFP and mCherry::RAB-5 at the cortex of <i>control(RNAi)</i> embryos during the polarity maintenance phase. The box is magnified three-fold in inset. Fluorescence intensity was measured along the line in each inset and represented for PAR-6 (green) or RAB-5 (red). The arrow points to a region with peaks of fluorescence intensity for each channel, and thus co-localization for both markers. (<b>B</b>) Images of PAR-6::mCherry and GFP::CHC-1 at the cortex of <i>control(RNAi)</i>, <i>rab-5(RNAi)</i>, <i>dyn-1(RNAi)</i> and <i>rab-5(RNAi); dyn-1(RNAi)</i> embryos during the polarity maintenance phase. Graphical representations are as in panel A, except that PAR-6 is in red and CHC-1 is in green. The bar graph shows that co-localization of PAR-6::mCherry with GFP::CHC-1 is significantly increased in <i>rab-5(RNAi)</i> embryos compared to <i>control(RNAi)</i> (red star, p = 0.022, Student's t-test) but not in <i>dyn-1(RNAi)</i> (p = 0.7) or in <i>rab-5(RNAi); dyn-1(RNAi)</i> (p = 0.89). The gray bars represent random co-localization control. n, number of puncta analyzed. Bars represent standard error of the mean. (<b>C</b>) Midplane images of PAR-6::GFP and mCherry::RAB-5 in the cytoplasm of <i>control(RNAi)</i> embryos during the polarity maintenance phase. Graphic representations are as in panel A. In all panels, anterior is to the left. Scale bars, 10 µm.</p

RAB-5 controls PAR-6 cortical localization independently of actomyosin contractility.

<p>(<b>A</b>) Midplane images of PAR-6::GFP in <i>control(RNAi)</i> and <i>rab-5(RNAi)</i> embryos at pronuclear meeting, i.e., at the end of the establishment phase of polarity. Quantitation of fluorescence intensity along the circumference of the cortex reveals that the size of PAR-6::GFP cortical domain is more anterior in <i>rab-5(RNAi)</i> embryos compared to <i>control(RNAi)</i> (red star, p = 6.11×10⁻⁰⁶, Student's t-test). (<b>B</b>) Midplane images of GFP::PAR-2 in <i>control(RNAi)</i> and <i>rab-5(RNAi)</i> embryos at pronuclear meeting. Quantitation of fluorescence intensity along the cortex shows that the size of GFP::PAR-2 cortical domain is similar in <i>control(RNAi)</i> and <i>rab-5(RNAi)</i> embryos (p = 0.98, Student's t-test). Quantitation excluded the anterior crescent of GFP::PAR-2 visible in wild-type embryos (arrow) but absent from <i>rab-5(RNAi</i>) embryos. (<b>C</b>) Images of NMY-2::GFP at the mid-plane of <i>control(RNAi)</i> and <i>rab-5(RNAi)</i> embryos at pronuclear meeting. Quantitation of fluorescence intensity along the cortex shows that the size of NMY-2::GFP cortical domain is similar in <i>control(RNAi)</i> and <i>rab-5(RNAi)</i> embryos (p = 0.17, Student's t-test). (<b>D</b>) Images of NMY-2::GFP at the cortex of <i>control(RNAi)</i> and <i>rab-5(RNAi)</i> embryos during the phase of establishment of polarity. Kymographs were generated along the antero-posterior axis (orange lines). The average velocity of NMY-2 puncta is similar in <i>control(RNAi)</i> and <i>rab-5(RNAi)</i> embryos (p = 0.86, Student's t-test). In each frame anterior is to the left and white arrowheads indicate the boundary of cortical fluorescence. n, number of embryos analyzed. Error bars represent standard deviation. Scale bars, 10 µm.</p

RAB-5 controls actin and PAR-6 organization at the cortex.

<p>(<b>A</b>) Images of dMoe::GFP at the cortex of <i>control(RNAi)</i>, <i>rab-5(RNAi)</i>, <i>dyn-1(RNAi)</i> and <i>rab-5(RNAi); dyn-1 (RNAi)</i> embryos during the phase of maintenance of polarity. Regions of low fluorescence intensity (LFI) are visible in all backgrounds (white arrows), but quantitation reveals that they are more frequent in <i>rab-5(RNAi)</i> and <i>rab-5(RNAi); dyn-1(RNAi)</i> embryos (red star, compared to <i>control(RNAi)</i>: p = 0.00027 and p = 0.0054 respectively, Student's t-test). n, number of embryos analyzed. Error bars represent standard error of the mean. (<b>B</b>) Images of dMoe::GFP at the cortex of <i>control(RNAi)</i> and <i>rab-5(RNAi)</i> embryos during the phase of maintenance of polarity. Timed quantitation by kymograph analysis shows that the LFI regions are visible for a longer time in <i>rab-5(RNAi)</i> embryos (red star, p = 0.037, Student's t-test). n, number of embryos analyzed. Error bars represent standard error of the mean. (<b>C</b>) Images of PAR-6::mCherry and dMoe::GFP at the cortex of <i>control(RNAi)</i> and <i>rab-5(RNAi)</i> embryos during the phase of maintenance of polarity. The box is magnified four-fold in inset. Fluorescence intensity was measured along the line in each inset and represented for PAR-6 (red) or dMoe (green). The black arrow in the graph points to a region 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 <i>rab-5(RNAi)</i> embryos compared to <i>control(RNAi)</i> (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. (<b>D</b>) Model depicting the regulation of PAR-6 function by RAB-5 (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035286#s3" target="_blank">discussion</a> for details).</p

Depletion of RAB-5 and other endocytic regulators results in polarity phenotypes in early <i>C. elegans</i> embryos.

<p>(<b>A</b>) Midplane DIC images from time-lapse acquisitions of developing wild-type or <i>par-2(it5ts)</i> embryos undergoing first or second division with the indicated RNAi treatment. All of the embryos depicted successfully completed their first and second cytokineses. In each frame, anterior is to the left and arrowheads indicate centrosome position. Scale bar, 10 µm. (<b>B</b>) Quantitation of posterior spindle displacement in zygotes of each background, as determined by measuring the ratio between the position of anterior vs posterior centrosome. (<b>C</b>) Quantitation of the asynchrony between AB and P₁ division at the 2-cell stage, as determined by measuring the time difference between cytokinesis onset in each cell. Stars indicate statistical significance (p<0.05, Student's t-test) when compared to <i>control(RNAi)</i> in wild type (red stars) or <i>par-2(it5ts)</i> backgrounds (blue stars). Depleting RAB-5 modulates all polarity-related phenotypes that were quantitated. n, number of embryos analyzed. Error bars represent standard deviation.</p

RAB-5 controls PAR-6 residence time at the cortex.

<p>(<b>A</b>) Time lapse images of PAR-6::GFP in the middle plane and cortical plane of <i>control(RNAi)</i> embryos. Imaging at the cortical plane was done by TIRF microscopy. A schematic representation of PAR protein localization in each plane is depicted on the left. (<b>B</b>) Magnified images of PAR-6::GFP at the cortex of <i>control(RNAi)</i> embryos obtained by TIRF microscopy. The white arrow points to a PAR-6-positive puncta appearing and disappearing from the focal plane during this 9-second excerpt. (<b>C</b>) TIRF images of cortical PAR-6::GFP in a <i>control(RNAi)</i> embryo before (upper image) and after (lower image) processing by particle tracking software. Each red dot on the bottom image is recognized as a PAR-6-positive structure. Quantitative automated analysis of PAR-6::GFP puncta shows that they have short cortical residence time during both the establishment and maintenance phases of polarity. The mean cortical residence time is significantly longer during the establishment phase than during the maintenance phase (red star, p = 0.003, Student's t-test). Error bars represent standard deviation. (<b>D</b>) TIRF images of cortical PAR-6::GFP in <i>rab-5(RNAi)</i> embryos. Quantitative automated analysis of PAR-6::GFP puncta shows that depletion of RAB-5 results in a significant increase in their mean cortical residence time during the maintenance phase (red star, p = 0.026, Student's t-test), but not during the establishment phase of polarity. Error bars represent standard deviation. White arrows point to cortical regions where PAR-6::GFP is excluded. In all panels anterior is to the left. Scale bars, 10 µm.</p