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 in vivo, and demonstrate a common mechanism that directs anaphase cell elongation and cleavage furrow progression during cell division. We show that onion rings (onr) and funnel cakes (fun) encode the Drosophila homologs of the Exo84 and Sec8 exocyst subunits, respectively. In onr and fun 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 onr and fun 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 onr and fun 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 onr and fun 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.
References
[1]
Prekeris R, Gould GW. Breaking up is hard to do-membrane traffic in cytokinesis. J Cell Sci. 2008;121: 1569–1576. doi: 10.1242/jcs.018770. pmid:18469013
[2]
Neto H, Collins LL, Gould GW. Vesicle trafficking and membrane remodelling in cytokinesis. Biochem J. 2011;437: 13–24. doi: 10.1042/BJ20110153. pmid:21668412
[3]
D'Avino PP, Giansanti MG, Petronczki M. Cytokinesis in animal cells. Cold Spring Harb Perspect Biol. 2015 Feb 13. pii: a015834. doi: 10.1101/cshperspect.a015834. pmid:25680833
[4]
Giansanti MG, Farkas RM, Bonaccorsi S, Lindsley DL, Wakimoto BT, Fuller MT, et al. Genetic dissection of meiotic cytokinesis in Drosophila males. Mol Biol Cell 2004;15: 2509–2522. pmid:15004238 doi: 10.1091/mbc.e03-08-0603
[5]
Brill JA, Hime GR, Scharer-Schuksz M, Fuller MT. A phospholipid kinase regulates actin organization and intercellular bridge formation during germline cytokinesis. Development 2000;127: 3855–3864. pmid:10934029
[6]
Xu H, Brill JA, Hsien J, McBride R, Boulianne GL, Trimble WS. Syntaxin 5 is required for cytokinesis and spermatid differentiation in Drosophila. Dev Biol. 2002;251: 294–306. pmid:12435359 doi: 10.1006/dbio.2002.0830
[7]
Farkas RM, Giansanti MG, Gatti M, Fuller MT. The Drosophila Cog5 homologue is required for cytokinesis, cell elongation, and assembly of specialized Golgi architecture during spermatogenesis. Mol Biol Cell. 2003;14: 190–200. pmid:12529436 doi: 10.1091/mbc.e02-06-0343
[8]
Dyer N, Rebollo E, Dominguez P, Elkhatib N, Chavrier P, Daviet L, et al. Spermatocyte cytokinesis requires rapid membrane addition mediated by ARF6 on central spindle recycling endosomes. Development 2007;134: 4437–4447. pmid:18039970 doi: 10.1242/dev.010983
[9]
Gatt MK, Glover DM. The Drosophila phosphatidylinositol transfer protein encoded by vibrator is essential to maintain cleavage-furrow ingression in cytokinesis. J Cell Sci. 2006;119: 2225–2235. pmid:16684816 doi: 10.1242/jcs.02933
[10]
Giansanti MG, Belloni G, Gatti M. Rab11 is required for membrane trafficking and actomyosin ring constriction in meiotic cytokinesis of Drosophila males. Mol Biol Cell. 2007;18: 5034–47. pmid:17914057 doi: 10.1091/mbc.e07-05-0415
[11]
Robinett CC, Giansanti MG, Gatti M, Fuller MT. TRAPPII is required for cleavage furrow ingression and localization of Rab11 in dividing male meiotic cells of Drosophila. J Cell Sci. 2009;122: 4526–4534. doi: 10.1242/jcs.054536. pmid:19934220
[12]
Belloni G, Sechi S, Riparbelli MG, Fuller MT, Callaini G, Giansanti MG. Mutations in Cog7 affect Golgi structure, meiotic cytokinesis and sperm development during Drosophila spermatogenesis. J Cell Sci. 2012;125: 5441–5452. doi: 10.1242/jcs.108878. pmid:22946051
[13]
Sechi S, Colotti G, Belloni G, Mattei V, Frappaolo A, Raffa GD, et al. GOLPH3 is essential for contractile ring formation and Rab11 localization to the cleavage site during cytokinesis in Drosophila melanogaster. PLoS Genet. 2014;10: e1004305. doi: 10.1371/journal.pgen.1004305. pmid:24786584
[14]
Sechi S, Frappaolo A, Belloni G, Colotti G, Giansanti MG. The multiple cellular functions of the oncoprotein Golgi phosphoprotein 3. Oncotarget 2015;6: 3493–3506. pmid:25691054 doi: 10.18632/oncotarget.3051
[15]
Lipschutz JH, Mostov KE. Exocytosis: the many masters of the exocyst. Curr Biol. 2002;12: 212–214. doi: 10.1016/s0960-9822(02)00753-4
[16]
Whyte JR, Munro S. Vesicle tethering complexes in membrane traffic. J Cell Sci. 2002;115: 2627–2637. pmid:12077354
[17]
Novick P, Field C, Schekman R. Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell 1980;21: 205–215. pmid:6996832 doi: 10.1016/0092-8674(80)90128-2
[18]
TerBush DR, Novick P. Sec6, Sec8, and Sec15 are components of a multisubunit complex which localizes to small bud tips in Saccharomyces cerevisiae. J Cell Biol. 1995;130: 299–312. pmid:7615633 doi: 10.1083/jcb.130.2.299
[19]
TerBush DR, Maurice T, Roth D, Novick P. The Exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae. EMBO J. 1996;15: 6483–6494. pmid:8978675
[20]
Kee Y, Yoo JS, Hazuka CD, Peterson KE, Hsu SC, Scheller RH. Subunit structure of the mammalian exocyst complex. Proc Natl Acad Sci USA 1997;94: 14438–14443. pmid:9405631 doi: 10.1073/pnas.94.26.14438
[21]
Grindstaff KK, Yeaman C, Anandasabapathy N, Hsu SC, Rodriguez-Boulan E, Scheller RH, et al. Sec6/8 complex is recruited to cell-cell contacts and specifies transport vesicle delivery to the basal-lateral membrane in epithelial cells. Cell 1998;93: 731–740. pmid:9630218 doi: 10.1016/s0092-8674(00)81435-x
[22]
Guo W, Grant A, Novick P. Exo84p is an exocyst protein essential for secretion. J Biol Chem. 1999;274: 23558–23564. pmid:10438536 doi: 10.1074/jbc.274.33.23558
[23]
Hsu SC, Hazuka CD, Foletti DL, Scheller RH. Targeting vesicles to specific sites on the plasma membrane: the role of the sec6/8 complex. Trends Cell Biol. 1999;9: 150–153. pmid:10203793 doi: 10.1016/s0962-8924(99)01516-0
[24]
Blankenship JT, Fuller MT, Zallen JA. The Drosophila homolog of the Exo84 exocyst subunit promotes apical epithelial identity. J Cell Sci. 2007;120: 3099–3110. pmid:17698923 doi: 10.1242/jcs.004770
[25]
Murthy M, Garza D, Scheller RH, Schwarz TL. Mutations in the exocyst component Sec5 disrupt neuronal membrane traffic, but neurotransmitter release persists. Neuron 2003;37: 433–447. pmid:12575951 doi: 10.1016/s0896-6273(03)00031-x
[26]
Murthy M, Schwarz TL. The exocyst component Sec5 is required for membrane traffic and polarity in the Drosophila ovary. Development. 2004;131: 377–388. pmid:14681190 doi: 10.1242/dev.00931
[27]
Murthy M, Schwarz TL. The exocyst component Sec5 is required for membrane traffic and polarity in the Drosophila ovary. Development. 2004;131: 377–388. pmid:14681190 doi: 10.1242/dev.00931
[28]
Sommer B, Oprins A, Rabouille C, Munro S. The exocyst component Sec5 is present on endocytic vesicles in the oocyte of Drosophila melanogaster. J Cell Biol. 2005;169: 953–963. pmid:15955846 doi: 10.1083/jcb.200411053
[29]
Jafar-Nejad H, Andrews HK, Acar M, Bayat V, Wirtz-Peitz F, Mehta SQ, et al. Sec15, a component of the exocyst, promotes notch signaling during the asymmetric division of Drosophila sensory organ precursors. Dev Cell 2005;9: 351–363. pmid:16137928 doi: 10.1016/j.devcel.2005.06.010
[30]
Mehta SQ, Hiesinger PR, Beronja S, Zhai RG, Schulze KL, Verstreken P, et al. Mutations in Drosophila sec15 reveal a function in neuronal targeting for a subset of exocyst components. Neuron 2005;46: 219–232. pmid:15848801 doi: 10.1016/j.neuron.2005.02.029
[31]
Murthy M, Teodoro RO, Miller TP, Schwarz TL. Sec5, a member of the exocyst complex, mediates Drosophila embryo cellularization. Development 2010;137: 2773–2783. doi: 10.1242/dev.048330. pmid:20630948
[32]
Fielding AB, Schonteich E, Matheson J, Wilson G, Yu X, Hickson GR, et al. Rab11-FIP3 and FIP4 interact with Arf6 and the exocyst to control membrane traffic in cytokinesis. EMBO J. 2005;24: 3389–3399. pmid:16148947 doi: 10.1038/sj.emboj.7600803
[33]
Gromley A, Yeaman C, Rosa J, Redick S, Chen CT, Mirabelle S, et al. Centriolin anchoring of exocyst and SNARE complexes at the midbody is required for secretory-vesicle-mediated abscission. Cell 2005;123: 75–87. pmid:16213214 doi: 10.1016/j.cell.2005.07.027
[34]
Chen XW, Inoue M, Hsu SC, Saltiel AR. RalA-exocyst-dependent recycling endosome trafficking is required for the completion of cytokinesis. J Biol Chem. 2006;281: 38609–38616. pmid:17028198 doi: 10.1074/jbc.m512847200
[35]
Cascone I, Selimoglu R, Ozdemir C, Del Nery E, Yeaman C, White M, et al. Distinct roles of RalA and RalB in the progression of cytokinesis are supported by distinct RalGEFs. EMBO J. 2008;27: 2375–2387. doi: 10.1038/emboj.2008.166. pmid:18756269
[36]
Royou A, Field C, Sisson JC, Sullivan W, Karess R. Reassessing the role and dynamics of nonmuscle Myosin II during furrow formation in early Drosophila Embryos. Mol Biol Cell 2004;15: 838–850. pmid:14657248 doi: 10.1091/mbc.e03-06-0440
[37]
Wong R, Hadjiyanni I, Wei HC, Polevoy G, McBride R, Sem KP,et al. PIP2 hydrolysis and calcium release are required for cytokinesis in Drosophila spermatocytes. Curr Biol. 2005;15: 1401–1406. pmid:16085493 doi: 10.1016/j.cub.2005.06.060
[38]
Inoue YH, Savoian MS, Suzuki T, Mathe E, Yamamoto MT, Glover DM. Mutations in orbit/mast reveal that the central spindle is comprised of two microtubule populations, those that initiate cleavage and those that propagate furrow ingression. J. Cell Biol. 2004;166: 49–60. pmid:15240569 doi: 10.1083/jcb.200402052
[39]
Wainman A, Giansanti MG, Goldberg ML, Gatti M. The Drosophila RZZ complex—roles in membrane trafficking and cytokinesis. J Cell Sci. 2012;125: 4014–4025. doi: 10.1242/jcs.099820. pmid:22685323
[40]
Sisson JC, Field C, Ventura R, Royou A, Sullivan W. Lava lamp, a novel peripheral golgi protein, is required for Drosophila melanogaster cellularization. J Cell Biol. 2000;151: 905–918. pmid:11076973 doi: 10.1083/jcb.151.4.905
[41]
Giansanti MG, Bonaccorsi S, Kurek R, Farkas RM, Dimitri P, Fuller MT, et al. The class I PITP giotto is required for Drosophila cytokinesis. Curr Biol. 2006;16: 195–201. pmid:16431372 doi: 10.1016/j.cub.2005.12.011
[42]
Yu IM, Hughson FM. Tethering factors as organizers of intracellular vesicular traffic. Annu Rev Cell Dev Biol. 2010;26: 137–156. doi: 10.1146/annurev.cellbio.042308.113327. pmid:19575650
[43]
Heider MR, Munson M. Exorcising the exocyst complex. Traffic 2012;13: 898–907. doi: 10.1111/j.1600-0854.2012.01353.x. pmid:22420621
[44]
Wang S, Liu Y, Adamson CL, Valdez G, Guo W, Hsu SC. The mammalian exocyst, a complex required for exocytosis, inhibits tubulin polymerization. J Biol Chem. 2004;279: 35958–66. pmid:15205466 doi: 10.1074/jbc.m313778200
Gerald NJ, Damer CK, O’Halloran TJ, De Lozanne A. Cytokinesis failure in clathrin-minus cells is caused by cleavage furrow instability. Cell Motil Cytoskeleton 2001;48: 213–223. pmid:11223952 doi: 10.1002/1097-0169(200103)48:3<213::aid-cm1010>3.0.co;2-v
[47]
Somma MP, Fasulo B, Cenci G, Cundari E, Gatti M. Molecular dissection of cytokinesis by RNA interference in Drosophila cutured cells. Mol Biol Cell 2002;13: 2448–2460. pmid:12134082 doi: 10.1091/mbc.01-12-0589
[48]
Giansanti MG, Fuller MT. What Drosophila spermatocytes tell us about the mechanisms underlying cytokinesis. Cytoskeleton 2012;69: 869–81 doi: 10.1002/cm.21063. pmid:22927345
[49]
Albertson R, Cao J, Hsieh TS, Sullivan W. Vesicles and actin are targeted to the cleavage furrow via furrow microtubules and the central spindle. J Cell Biol. 2008;181: 777–790. doi: 10.1083/jcb.200803096. pmid:18504302
[50]
Zhang XM, Ellis S, Sriratana A, Mitchell CA, Rowe T. Sec15 is an effector for the Rab11 GTPase in mammalian cells. J Biol Chem. 2004;279: 43027–43034. pmid:15292201 doi: 10.1074/jbc.m402264200
[51]
Beronja S, Laprise P, Papoulas O, Pellikka M, Sisson J, Tepass U. Essential function of Drosophila Sec6 in apical exocytosis of epithelial photoreceptor cells. J Cell Biol. 2005;169: 635–46. pmid:15897260 doi: 10.1083/jcb.200410081
[52]
Lanjevin J, Morgan MJ, Sibarita JB, Aresta S, Murthy M, Schwarz T, et al. Drosophila exocyst components Sec5, Sec6, and Sec15 regulate DE-Cadherin trafficking from recycling endosomes to the plasma membrane. Dev Biol. 2005;9: 365–376. doi: 10.1016/j.devcel.2005.07.013
[53]
Wu S, Mehta SQ, Pichaud F, Bellen HJ, Quiocho FA. Sec15 interacts with Rab11 via a novel domain and affects Rab11 localization in vivo. Nat Struct Mol Biol. 2005;12: 879–885. pmid:16155582 doi: 10.1038/nsmb987
[54]
Rivera-Molina F, Toomre D. Live-cell imaging of exocyst links its spatiotemporal dynamics to various stages of vesicle fusion. J Cell Biol. 2013;201: 673–680. doi: 10.1083/jcb.201212103. pmid:23690179
[55]
Hara Y, Kimura A. Cell-size-dependent spindle elongation in the Caenorhabditis elegans early embryo. Current Biology 2009;19: 1549–1554. doi: 10.1016/j.cub.2009.07.050. pmid:19682904
[56]
Kotadia S, Montembault E, Sullivan W, Royou A. Cell elongation is an adaptive response for clearing long chromatid arms from the cleavage plane. J Cell Biol. 2012;199: 745–53. doi: 10.1083/jcb.201208041. pmid:23185030
[57]
Hickson GRX, Echard A, O’Farrell PH. Rho-kinase Controls Cell Shape Changes during Cytokinesis. Curr Biol. 2006;16: 359–370. pmid:16488869 doi: 10.1016/j.cub.2005.12.043
[58]
Rodrigues NT, Lekomtsev S, Jananji S, Kriston-Vizi J, Hickson GR, Baum B. Kinetochore-localized PP1-Sds22 couples chromosome segregation to polar relaxation. Nature 2015; doi: 10.1038/nature14496 [Epub ahead of print].
[59]
Albertson R, Riggs B, Sullivan W Membrane traffic: a driving force in cytokinesis. Trends Cell Biol. 2005;15: 92–101. pmid:15695096 doi: 10.1016/j.tcb.2004.12.008
[60]
McKay HF, Burgess DR. 'Life is a highway': membrane trafficking during cytokinesis. Traffic 2011;12: 247–251. doi: 10.1111/j.1600-0854.2010.01139.x. pmid:21054718
[61]
Hehnly H, Doxsey S. Rab11 endosomes contribute to mitotic spindle organization and orientation. Dev Cell. 2014; 28:497–507. doi: 10.1016/j.devcel.2014.01.014. pmid:24561039
[62]
Fendrych M, Synek L, Pecenková T, Toupalová H, Cole R, Drdová E, Nebesárová J, Sedinová M, Hála M, Fowler JE, Zársky V. The Arabidopsis exocyst complex is involved in cytokinesis and cell plate maturation. Plant Cell. 2010;22:3053–65. doi: 10.1105/tpc.110.074351. pmid:20870962
[63]
Wu J1, Tan X, Wu C, Cao K, Li Y, Bao Y. Regulation of cytokinesis by exocyst subunit SEC6 and KEULE in Arabidopsis thaliana. Mol Plant. 2013;6:1863–76. doi: 10.1093/mp/sst082. pmid:23702595
[64]
Rybak K, Steiner A, Synek L, Klaeger S, Kulich I, Facher E, Wanner G, Kuster B, Zarsky V, Persson S, Assaad FF. Plant cytokinesis is orchestrated by the sequential action of the TRAPPII and exocyst tethering complexes. Dev Cell. 2014; 29:607–20. doi: 10.1016/j.devcel.2014.04.029. pmid:24882377
[65]
Dollar G, Struckhoff E, Michaud J, Cohen RS. Rab11 polarization of the Drosophila oocyte: a novel link between membrane trafficking, microtubule organization, and oskar mRNA localization and translation. Development 2002;129: 517–526. pmid:11807042
[66]
Zhang J, Schulze KL, Hiesinger PR, Suyama K, Wang S, Fish M, et al. Thirty-one flavors of Drosophila Rab proteins. 2007;176: 1307–1322.
[67]
Chen D, McKearin DM. A discrete transcriptional silencer in the bam gene determines asymmetric division of the Drosophila germline stem cell. Development 2003;130: 1159–1170. pmid:12571107 doi: 10.1242/dev.00325
Goldbach P, Wong R, Beise N, Sarpal R, Trimble WS, Brill JA. Stabilization of the actomyosin ring enables spermatocyte cytokinesis in Drosophila. Mol Biol Cell. 2010;21:1482–1493. doi: 10.1091/mbc.E09-08-0714. pmid:20237160
