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 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

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 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

Exocyst-Dependent Membrane Addition Is Required for Anaphase Cell Elongation and Cytokinesis in <i>Drosophila</i>

<div><p>Mitotic and cytokinetic processes harness cell machinery to drive chromosomal segregation and the physical separation of dividing cells. Here, we investigate the functional requirements for exocyst complex function during cell division <i>in vivo</i>, and demonstrate a common mechanism that directs anaphase cell elongation and cleavage furrow progression during cell division. We show that <i>onion rings (onr)</i> and <i>funnel cakes (fun)</i> encode the <i>Drosophila</i> homologs of the Exo84 and Sec8 exocyst subunits, respectively. In <i>onr</i> and <i>fun</i> mutant cells, contractile ring proteins are recruited to the equatorial region of dividing spermatocytes. However, cytokinesis is disrupted early in furrow ingression, leading to cytokinesis failure. We use high temporal and spatial resolution confocal imaging with automated computational analysis to quantitatively compare wild-type versus <i>onr</i> and <i>fun</i> mutant cells. These results demonstrate that anaphase cell elongation is grossly disrupted in cells that are compromised in exocyst complex function. Additionally, we observe that the increase in cell surface area in wild type peaks a few minutes into cytokinesis, and that <i>onr</i> and <i>fun</i> mutant cells have a greatly reduced rate of surface area growth specifically during cell division. Analysis by transmission electron microscopy reveals a massive build-up of cytoplasmic astral membrane and loss of normal Golgi architecture in <i>onr</i> and <i>fun</i> spermatocytes, suggesting that exocyst complex is required for proper vesicular trafficking through these compartments. Moreover, recruitment of the small GTPase Rab11 and the PITP Giotto to the cleavage site depends on wild-type function of the exocyst subunits Exo84 and Sec8. Finally, we show that the exocyst subunit Sec5 coimmunoprecipitates with Rab11. Our results are consistent with the exocyst complex mediating an essential, coordinated increase in cell surface area that potentiates anaphase cell elongation and cleavage furrow ingression.</p></div

<i>onr</i> and <i>fun</i> mutations interact with mutations in <i>Rab11</i>.

<p>(A) Frequencies of early spermatids containing 2, 4 or more than 4 nuclei per nebenkern in testes from either <i>Rab11</i>^<i>93Bi</i><i>/Rab11</i>^<i>93Bi</i><i>(Rab11) or fun</i>^<i>z1010</i><i>Rab11</i>^<i>93Bi</i><i>/+ Rab11</i>^<i>93Bi</i><i>(fun Rab11/Rab11)</i> mutant males. (B) Frequencies of early spermatids containing multiple nuclei (2, 4 or more than 4 nuclei) per nebenkern in testes from either <i>Rab11</i>^<i>93Bi</i>/<i>Rab11</i>^{<i>E(To)3</i>}<i>(Rab11)</i>, <i>fun</i>^<i>z1010</i><i>/fun</i>^<i>z1010</i><i>(fun)</i>, or <i>fun</i>^<i>z1010</i><i>Rab11</i>^<i>93Bi</i><i>fun</i>^<i>z1010</i><i>Rab11</i>^{<i>E(To)3</i>}<i>(fun Rab11)</i> mutant males. (C) Co-IP of HA-Sec8 with GFP-Exo84. Protein extracts from testes expressing either HA-Sec8 and GFP-Exo84 or HA-Sec8 alone were immunoprecipitated with anti-GFP (i.e., GFP-trap beads) and immunoblotted for either GFP, HA or Rab11. (D) Co-IP of Sec5 with YFP-Rab11. Protein extracts from testes expressing either wild-type YFP-Rab11 (wt), YFP-Rab11^Q70L (Q70L) or YFP-Rab11^S25N (S25N) were immunoprecipitated for YFP (using GFP-trap beads) and blotted for either YFP or Sec5.</p

Localization of exocyst complex proteins in dividing spermatocytes.

<p>(A) Localization of Sec8 protein in wild-type primary spermatocytes. Interphase and dividing spermatocytes were stained for Tubulin (green), Sec8 (red) and DNA (blue). During interphase, Sec8 was mostly diffuse in the cytoplasm and enriched at the plasma membrane (arrowheads). In dividing spermatocytes, Sec8 appeared enriched in a broad cortical band that encircled the midzone (arrows) and was excluded from the poles. (B) Localization of Sec5 protein in wild-type dividing spermatocytes. Primary spermatocytes were stained for Tubulin (green), Sec5 (red) and DNA (blue). Note the enrichment of Sec5 in puncta at the astral microtubules (arrowhead) and at the cleavage furrow (arrows). Scale bar, 10 μm.</p

Defective cytokinetic ring ingression in <i>fun</i> and <i>onr</i> mutant cells.

<p>(A) Selected still frames from supplemental <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005632#pgen.1005632.s005" target="_blank">S1</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005632#pgen.1005632.s006" target="_blank">S2</a></b>and <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005632#pgen.1005632.s007" target="_blank">S3</a></b>Movies. Dividing spermatocytes expressing the regulatory light chain of non-muscle myosin II, Sqh-GFP, were imaged starting from the beginning of anaphase. Numbers at the bottom of each frame indicate minutes from the beginning of imaging. Note that the Sqh-GFP ring undergoes minimal constriction (<i>fun</i>) or fails to constrict (<i>onr</i>) in mutant cells. Scale bar, 10μm. (B) Dynamics of cleavage furrows in <i>fun</i> and <i>onr</i> mutants. Furrow diameters (relative to the diameter at t = 0) in dividing spermatocytes from wild type, <i>fun</i>^<i>z1010</i><i>/Df(3R)Exel6145</i> (<i>fun</i>) and <i>onr</i>^<i>z4840</i><i>/Df(3R)Espl1 (onr)</i> males expressing Sqh-GFP and undergoing ana-telophase were plotted over time. (C) Furrow diameters (relative to the diameter at time = 0) were plotted at 5-minute intervals. Furrow diameters were measured in movies from dividing spermatocytes expressing Sqh-GFP and undergoing ana-telophases (n = 9 wild type, n = 8 <i>fun</i> and n = 8 <i>onr)</i>. Error bars indicate standard deviations. *p = 0.0035, **p = 0.0008;***p = 0.0001, significantly different from control in the Student t test.</p

Defects in morphology and ultrastructure of parafusorial membranes and Golgi bodies in <i>fun</i> and <i>onr</i> mutant cells.

<p>Transmission electron micrographs showing parafusorial membranes (A-F), astral membranes (G-I), and Golgi bodies (J-L) in <i>fun</i> and <i>onr</i> mutant spermatocytes. Parafusorial and astral membranes (arrows) are enlarged, fragmented and vacuolated in <i>fun</i>^<i>z1010</i>/<i>Df(3R)Exel6145</i> (B, E, H) and <i>onr</i>^<i>z4840</i><i>/Df(3R)Espl3</i> (C, F, I) dividing spermatocytes. (D, E, F) panels are magnified images of areas surrounded by white squares in (A, B, C). (H, I) panels are magnified images of areas surrounded by black squares in (B, C). Golgi bodies (asterisks) show vacuolated regions in <i>fun</i> (K) and <i>onr</i> (L) mutant spermatocytes. Golgi bodies surrounded by white squares in (J-L) are magnified in insets. Scale bars are 2 μm (A-C, J, K) or 500 nm (D-I, L).</p