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

The PITP Giotto fails to concentrate at the midzone of dividing spermatocytes from <i>fun</i> and <i>onr</i> males.

<p>Spermatocytes were stained with anti-Tubulin (green), anti-Gio (red) and DAPI (blue). Arrows indicate the cleavage site. Wild type, <i>fun</i>^<i>z1010</i>/<i>Df(3R)Exel6145</i>, and <i>onr</i>^<i>z4840</i><i>/Df(3R)Espl3</i> were stained for Tubulin (green), Gio (red) and DNA (Blue). Double arrowheads point to astral membranes, arrowheads indicate parafusorial membranes. Scale bar, 10 μm.</p

Failure in anaphase elongation, cleavage furrow progression, and surface area addition in <i>onr</i> and <i>fun</i> mutant cells.

<p>(A-C) Still frames from time-lapse confocal microscopy of wild-type (A), <i>onr</i>^<i>z4840</i>mutant (B), and <i>fun</i>^<i>z1010</i> mutant (C) male germline cells expressing PLCδd-PH-GFP and β-Tub-GFP (imaged simultaneously in single channel). Cells are shown just prior to elongation (A, B, C), immediately before ingression (A’, B’, C’), during ingression (A”, B”, C”), and after successfully completing or failing to complete cytokinesis (A”‘, B”‘, C”‘). (D-F) Representative segmented and voxelized cells of wild-type (D), <i>onr</i>^<i>z4840</i> mutant (E), and <i>fun</i>^<i>z1010</i> mutant (F) cells. (G-J) Quantitative computational analysis of surface area (G), volume (H), aspect ratio (I), and convex hull volume ratio (a measurement of furrow ingression, J) in wild-type (blue), <i>onr</i>^<i>z4840</i> mutant (red), and <i>fun</i>^<i>z1010</i> mutant (green) cells. Left, lines are average values of wild-type (n = 8), <i>onr</i> (n = 11), and <i>fun</i> (n = 10) segmented cells. Data from individual cells were aligned such that t = 0 is the start of anaphase elongation, while arrowheads mark the initiation of cytokinesis in wild-type cells (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005632#sec011" target="_blank">Materials and Methods</a>). Right, quantitation of percent change from t = 0 to t = 25 min. Increases in surface area, aspect ratio, and CHVR observed in wild-type cells are disrupted in <i>onr</i>^<i>z4840</i> and <i>fun</i>^<i>z1010</i> mutant cells, while no significant difference is observed in volume. Prior to the start of anaphase elongation, cell volume and surface area were nearly identical in wild type, <i>onr</i> mutant, and <i>fun</i> mutant cells (p-values ranging from 0.0838 to 0.5969). All movies start during early anaphase and end after successful (wild-type) or failed (<i>fun</i> and <i>onr</i>) cytokinesis. Shaded region indicates standard error (G-J); *p<0.0001, significantly different from control in the two-sample Student t-test; n.s. = not significant, p>0.23. Scale bar, 10 μm.</p

Defects in Golgi structure in <i>fun</i> and <i>onr</i> mutant cells.

<p>(A) G2 primary spermatocytes from wild-type, <i>onr</i>^<i>z4840</i><i>/Df(3R)Espl3</i> and <i>fun</i>^<i>z1010</i>/<i>Df(3R)Exel6145</i> mutant males, stained for the Golgin Lva (red) and DNA (blue). Enlargements of Golgi stacks are shown on the right of each panel. Scale bar, 10 μm. (B) Average number of Golgi bodies per cell (± SEM) visualized in G2 spermatocytes from wild type (n = 50), <i>onr</i>^<i>z4840</i><i>/Df(3R)Espl3 (onr</i>, n = 48), or <i>fun</i>^<i>z1010</i>/<i>Df(3R)Exel614</i> (<i>fun</i>, N = 48) after staining for Lva. Numbers of Golgi per cell in <i>fun</i> and <i>onr</i> mutants are significantly different from wild type in the Student t test:*p<0.0001, **p<0.0001. (C) Average area (± SEM) of Golgi bodies, quantified by ImageJ (expressed in arbitrary units), in G2 primary spermatocytes stained for Lva, Golgi sizes are significantly different in <i>fun</i>^<i>z1010</i>/<i>Df(3R)Exel614</i> (<i>fun</i>) and <i>onr</i>^<i>z4840</i><i>/Df(3R)Espl3 (onr)</i> compared to wild type using the Student t test, *p<0.0001, **p<0.0001.</p