C. elegans CLASP/CLS-2 negatively regulates membrane ingression throughout the oocyte cortex and is required for polar body extrusion

Autoři: Aleesa J. Schlientz aff001;  Bruce Bowerman aff001
Působiště autorů: Institute of Molecular Biology, University of Oregon, Eugene, OR, United States of America aff001
Vyšlo v časopise: C. elegans CLASP/CLS-2 negatively regulates membrane ingression throughout the oocyte cortex and is required for polar body extrusion. PLoS Genet 16(10): e32767. doi:10.1371/journal.pgen.1008751
Kategorie: Research Article
doi: 10.1371/journal.pgen.1008751


The requirements for oocyte meiotic cytokinesis during polar body extrusion are not well understood. In particular, the relationship between the oocyte meiotic spindle and polar body contractile ring dynamics remains largely unknown. We have used live cell imaging and spindle assembly defective mutants lacking the function of CLASP/CLS-2, kinesin-12/KLP-18, or katanin/MEI-1 to investigate the relationship between meiotic spindle structure and polar body extrusion in C. elegans oocytes. We show that spindle bipolarity and chromosome segregation are not required for polar body contractile ring formation and chromosome extrusion in klp-18 mutants. In contrast, oocytes with similarly severe spindle assembly defects due to loss of CLS-2 or MEI-1 have penetrant and distinct polar body extrusion defects: CLS-2 is required early for contractile ring assembly or stability, while MEI-1 is required later for contractile ring constriction. We also show that CLS-2 both negatively regulates membrane ingression throughout the oocyte cortex during meiosis I, and influences the dynamics of the central spindle-associated proteins Aurora B/AIR-2 and MgcRacGAP/CYK-4. We suggest that proper regulation by CLS-2 of both oocyte cortical stiffness and central spindle protein dynamics may influence contractile ring assembly during polar body extrusion in C. elegans oocytes.

Klíčová slova:

Caenorhabditis elegans – Cell membranes – Cell polarity – Cellular extrusion – Chromosome structure and function – Meiosis – Microtubules – Oocytes


1. Dumont J, Desai A. Acentrosomal spindle assembly and chromosome segregation during oocyte meiosis. Trends Cell Biol. 2012;22(5):241–9. doi: 10.1016/j.tcb.2012.02.007 22480579

2. Severson AF, von Dassow G, Bowerman B. Oocyte Meiotic Spindle Assembly and Function. Curr Top Dev Biol. 2016;116:65–98. doi: 10.1016/bs.ctdb.2015.11.031 26970614

3. Mullen TJ, Davis-Roca AC, Wignall SM. Spindle assembly and chromosome dynamics during oocyte meiosis. Curr Opin Cell Biol. 2019;60:53–9. doi: 10.1016/j.ceb.2019.03.014 31082633

4. Ohkura H. Meiosis: an overview of key differences from mitosis. Cold Spring Harb Perspect Biol. 2015;7(5).

5. Maddox AS, Azoury J, Dumont J. Polar body cytokinesis. Cytoskeleton (Hoboken). 2012;69(11):855–68. doi: 10.1002/cm.21064 22927361

6. Fabritius AS, Flynn JR, McNally FJ. Initial diameter of the polar body contractile ring is minimized by the centralspindlin complex. Dev Biol. 2011;359(1):137–48. doi: 10.1016/j.ydbio.2011.08.013 21889938

7. Dorn JF, Zhang L, Paradis V, Edoh-Bedi D, Jusu S, Maddox PS, et al. Actomyosin tube formation in polar body cytokinesis requires Anillin in C. elegans. Curr Biol. 2010;20(22):2046–51. doi: 10.1016/j.cub.2010.10.030 21055941

8. Pintard L, Bowerman B. Mitotic Cell Division in Caenorhabditis elegans. Genetics. 2019;211(1):35–73. doi: 10.1534/genetics.118.301367 30626640

9. Green RA, Paluch E, Oegema K. Cytokinesis in animal cells. Annu Rev Cell Dev Biol. 2012;28:29–58. doi: 10.1146/annurev-cellbio-101011-155718 22804577

10. Basant A, Glotzer M. Spatiotemporal Regulation of RhoA during Cytokinesis. Curr Biol. 2018;28(9):R570–R80. doi: 10.1016/j.cub.2018.03.045 29738735

11. Shelton CA, Carter JC, Ellis GC, Bowerman B. The nonmuscle myosin regulatory light chain gene mlc-4 is required for cytokinesis, anterior-posterior polarity, and body morphology during Caenorhabditis elegans embryogenesis. J Cell Biol. 1999;146(2):439–51. doi: 10.1083/jcb.146.2.439 10427096

12. Swan KA, Severson AF, Carter JC, Martin PR, Schnabel H, Schnabel R, et al. cyk-1: a C. elegans FH gene required for a late step in embryonic cytokinesis. J Cell Sci. 1998;111 (Pt 14):2017–27.

13. Schonegg S, Hyman AA. CDC-42 and RHO-1 coordinate acto-myosin contractility and PAR protein localization during polarity establishment in C. elegans embryos. Development. 2006;133(18):3507–16. doi: 10.1242/dev.02527 16899536

14. Schumacher JM, Golden A, Donovan PJ. AIR-2: An Aurora/Ipl1-related protein kinase associated with chromosomes and midbody microtubules is required for polar body extrusion and cytokinesis in Caenorhabditis elegans embryos. J Cell Biol. 1998;143(6):1635–46. doi: 10.1083/jcb.143.6.1635 9852156

15. Flynn JR, McNally FJ. A casein kinase 1 prevents expulsion of the oocyte meiotic spindle into a polar body by regulating cortical contractility. Mol Biol Cell. 2017;28(18):2410–9. doi: 10.1091/mbc.E17-01-0056 28701347

16. Gomes JE, Tavernier N, Richaudeau B, Formstecher E, Boulin T, Mains PE, et al. Microtubule severing by the katanin complex is activated by PPFR-1-dependent MEI-1 dephosphorylation. J Cell Biol. 2013;202(3):431–9. doi: 10.1083/jcb.201304174 23918937

17. Deng M, Li R. Sperm chromatin-induced ectopic polar body extrusion in mouse eggs after ICSI and delayed egg activation. PLoS One. 2009;4(9):e7171. doi: 10.1371/journal.pone.0007171 19787051

18. Deng M, Suraneni P, Schultz RM, Li R. The Ran GTPase mediates chromatin signaling to control cortical polarity during polar body extrusion in mouse oocytes. Dev Cell. 2007;12(2):301–8. doi: 10.1016/j.devcel.2006.11.008 17276346

19. Askjaer P, Galy V, Hannak E, Mattaj IW. Ran GTPase cycle and importins alpha and beta are essential for spindle formation and nuclear envelope assembly in living Caenorhabditis elegans embryos. Mol Biol Cell. 2002;13(12):4355–70. doi: 10.1091/mbc.e02-06-0346 12475958

20. Chuang CH, Schlientz AJ, Yang J, Bowerman B. Microtubule assembly and pole coalescence: early steps in C aenorhabditis elegans oocyte meiosis I spindle assembly. Biol Open. 2020;9(6).

21. Byrnes AE, Slep KC. TOG-tubulin binding specificity promotes microtubule dynamics and mitotic spindle formation. J Cell Biol. 2017;216(6):1641–57. doi: 10.1083/jcb.201610090 28512144

22. Patel K, Nogales E, Heald R. Multiple domains of human CLASP contribute to microtubule dynamics and organization in vitro and in Xenopus egg extracts. Cytoskeleton (Hoboken). 2012;69(3):155–65. doi: 10.1002/cm.21005 22278908

23. Al-Bassam J, Kim H, Brouhard G, van Oijen A, Harrison SC, Chang F. CLASP promotes microtubule rescue by recruiting tubulin dimers to the microtubule. Dev Cell. 2010;19(2):245–58. doi: 10.1016/j.devcel.2010.07.016 20708587

24. Aher A, Kok M, Sharma A, Rai A, Olieric N, Rodriguez-Garcia R, et al. CLASP Suppresses Microtubule Catastrophes through a Single TOG Domain. Dev Cell. 2018;46(1):40–58 e8. doi: 10.1016/j.devcel.2018.05.032 29937387

25. Tsvetkov AS, Samsonov A, Akhmanova A, Galjart N, Popov SV. Microtubule-binding proteins CLASP1 and CLASP2 interact with actin filaments. Cell Motil Cytoskeleton. 2007;64(7):519–30. doi: 10.1002/cm.20201 17342765

26. Dumont J, Oegema K, Desai A. A kinetochore-independent mechanism drives anaphase chromosome separation during acentrosomal meiosis. Nat Cell Biol. 2010;12(9):894–901. doi: 10.1038/ncb2093 20729837

27. Maton G, Edwards F, Lacroix B, Stefanutti M, Laband K, Lieury T, et al. Kinetochore components are required for central spindle assembly. Nat Cell Biol. 2015;17(7):953.

28. Espiritu EB, Krueger LE, Ye A, Rose LS. CLASPs function redundantly to regulate astral microtubules in the C. elegans embryo. Dev Biol. 2012;368(2):242–54. doi: 10.1016/j.ydbio.2012.05.016 22613359

29. Pelisch F, Bel Borja L, Jaffray EG, Hay RT. Sumoylation regulates protein dynamics during meiotic chromosome segregation in C. elegans oocytes. J Cell Sci. 2019;132(14).

30. Tanenbaum ME, Macurek L, Janssen A, Geers EF, Alvarez-Fernandez M, Medema RH. Kif15 cooperates with eg5 to promote bipolar spindle assembly. Curr Biol. 2009;19(20):1703–11. doi: 10.1016/j.cub.2009.08.027 19818618

31. Sturgill EG, Ohi R. Kinesin-12 differentially affects spindle assembly depending on its microtubule substrate. Curr Biol. 2013;23(14):1280–90. doi: 10.1016/j.cub.2013.05.043 23791727

32. Vanneste D, Takagi M, Imamoto N, Vernos I. The role of Hklp2 in the stabilization and maintenance of spindle bipolarity. Curr Biol. 2009;19(20):1712–7. doi: 10.1016/j.cub.2009.09.019 19818619

33. Connolly AA, Osterberg V, Christensen S, Price M, Lu C, Chicas-Cruz K, et al. Caenorhabditis elegans oocyte meiotic spindle pole assembly requires microtubule severing and the calponin homology domain protein ASPM-1. Mol Biol Cell. 2014;25(8):1298–311. doi: 10.1091/mbc.E13-11-0687 24554763

34. Wignall SM, Villeneuve AM. Lateral microtubule bundles promote chromosome alignment during acentrosomal oocyte meiosis. Nat Cell Biol. 2009;11(7):839–44. doi: 10.1038/ncb1891 19525937

35. Wolff ID, Tran MV, Mullen TJ, Villeneuve AM, Wignall SM. Assembly of Caenorhabditis elegans acentrosomal spindles occurs without evident microtubule-organizing centers and requires microtubule sorting by KLP-18/kinesin-12 and MESP-1. Mol Biol Cell. 2016;27(20):3122–31. doi: 10.1091/mbc.E16-05-0291 27559133

36. Clark-Maguire S, Mains PE. mei-1, a gene required for meiotic spindle formation in Caenorhabditis elegans, is a member of a family of ATPases. Genetics. 1994;136(2):533–46. 8150281

37. Srayko M, Buster DW, Bazirgan OA, McNally FJ, Mains PE. MEI-1/MEI-2 katanin-like microtubule severing activity is required for Caenorhabditis elegans meiosis. Genes Dev. 2000;14(9):1072–84. 10809666

38. McNally K, Berg E, Cortes DB, Hernandez V, Mains PE, McNally FJ. Katanin maintains meiotic metaphase chromosome alignment and spindle structure in vivo and has multiple effects on microtubules in vitro. Mol Biol Cell. 2014;25(7):1037–49. doi: 10.1091/mbc.E13-12-0764 24501424

39. Joly N, Martino L, Gigant E, Dumont J, Pintard L. Microtubule-severing activity of the AAA+ ATPase Katanin is essential for female meiotic spindle assembly. Development. 2016;143(19):3604–14. doi: 10.1242/dev.140830 27578779

40. Laband K, Le Borgne R, Edwards F, Stefanutti M, Canman JC, Verbavatz JM, et al. Chromosome segregation occurs by microtubule pushing in oocytes. Nat Commun. 2017;8(1):1499. doi: 10.1038/s41467-017-01539-8 29133801

41. Clandinin TR, Mains PE. Genetic studies of mei-1 gene activity during the transition from meiosis to mitosis in Caenorhabditis elegans. Genetics. 1993;134(1):199–210. 8514128

42. Mains PE, Kemphues KJ, Sprunger SA, Sulston IA, Wood WB. Mutations affecting the meiotic and mitotic divisions of the early Caenorhabditis elegans embryo. Genetics. 1990;126(3):593–605. 2249759

43. Han X, Gomes JE, Birmingham CL, Pintard L, Sugimoto A, Mains PE. The role of protein phosphatase 4 in regulating microtubule severing in the Caenorhabditis elegans embryo. Genetics. 2009;181(3):933–43. doi: 10.1534/genetics.108.096016 19087961

44. Monen J, Maddox PS, Hyndman F, Oegema K, Desai A. Differential role of CENP-A in the segregation of holocentric C. elegans chromosomes during meiosis and mitosis. Nat Cell Biol. 2005;7(12):1248–55. doi: 10.1038/ncb1331 16273096

45. Connolly AA, Sugioka K, Chuang CH, Lowry JB, Bowerman B. KLP-7 acts through the Ndc80 complex to limit pole number in C. elegans oocyte meiotic spindle assembly. J Cell Biol. 2015;210(6):917–32. doi: 10.1083/jcb.201412010 26370499

46. Piekny AJ, Maddox AS. The myriad roles of Anillin during cytokinesis. Semin Cell Dev Biol. 2010;21(9):881–91. doi: 10.1016/j.semcdb.2010.08.002 20732437

47. Davis-Roca AC, Muscat CC, Wignall SM. oocytes detect meiotic errors in the absence of canonical end-on kinetochore attachments. J Cell Biol. 2017;216(5):1243–53. doi: 10.1083/jcb.201608042 28356326

48. Gigant E, Stefanutti M, Laband K, Gluszek-Kustusz A, Edwards F, Lacroix B, et al. Inhibition of ectopic microtubule assembly by the kinesin-13 KLP-7 prevents chromosome segregation and cytokinesis defects in oocytes. Development. 2017;144(9):1674–86. doi: 10.1242/dev.147504 28289130

49. Canman JC, Cameron LA, Maddox PS, Straight A, Tirnauer JS, Mitchison TJ, et al. Determining the position of the cell division plane. Nature. 2003;424(6952):1074–8. doi: 10.1038/nature01860 12904818

50. Srayko M, O'Toole E T, Hyman AA, Muller-Reichert T. Katanin disrupts the microtubule lattice and increases polymer number in C. elegans meiosis. Curr Biol. 2006;16(19):1944–9. doi: 10.1016/j.cub.2006.08.029 17027492

51. 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(1):49–60. doi: 10.1083/jcb.200402052 15240569

52. Kitazawa D, Matsuo T, Kaizuka K, Miyauchi C, Hayashi D, Inoue YH. Orbit/CLASP is required for myosin accumulation at the cleavage furrow in Drosophila male meiosis. PLoS One. 2014;9(5):e93669. doi: 10.1371/journal.pone.0093669 24850412

53. Mimori-Kiyosue Y, Grigoriev I, Lansbergen G, Sasaki H, Matsui C, Severin F, et al. CLASP1 and CLASP2 bind to EB1 and regulate microtubule plus-end dynamics at the cell cortex. J Cell Biol. 2005;168(1):141–53. doi: 10.1083/jcb.200405094 15631994

54. Lansbergen G, Grigoriev I, Mimori-Kiyosue Y, Ohtsuka T, Higa S, Kitajima I, et al. CLASPs attach microtubule plus ends to the cell cortex through a complex with LL5beta. Dev Cell. 2006;11(1):21–32. doi: 10.1016/j.devcel.2006.05.012 16824950

55. Hagmann J, Burger MM, Dagan D. Regulation of plasma membrane blebbing by the cytoskeleton. J Cell Biochem. 1999;73(4):488–99. 10733343

56. Sugiyama T, Pramanik MK, Yumura S. Microtubule-Mediated Inositol Lipid Signaling Plays Critical Roles in Regulation of Blebbing. PLoS One. 2015;10(8):e0137032. doi: 10.1371/journal.pone.0137032 26317626

57. Tinevez JY, Schulze U, Salbreux G, Roensch J, Joanny JF, Paluch E. Role of cortical tension in bleb growth. Proc Natl Acad Sci U S A. 2009;106(44):18581–6. doi: 10.1073/pnas.0903353106 19846787

58. McNally KL, Martin JL, Ellefson M, McNally FJ. Kinesin-dependent transport results in polarized migration of the nucleus in oocytes and inward movement of yolk granules in meiotic embryos. Dev Biol. 2010;339(1):126–40. doi: 10.1016/j.ydbio.2009.12.021 20036653

59. Yang H-y, McNally K, McNally FJ. MEI-1/katanin is required for translocation of the meiosis I spindle to the oocyte cortex in C. elegans☆. Developmental Biology. 2003;260(1):245–59. doi: 10.1016/s0012-1606(03)00216-1 12885567

60. Vargas E, McNally KP, Cortes DB, Panzica MT, Danlasky BM, Li Q, et al. Spherical spindle shape promotes perpendicular cortical orientation by preventing isometric cortical pulling on both spindle poles during C. elegans female meiosis. Development. 2019;146(20).

61. Sumiyoshi E, Fukata Y, Namai S, Sugimoto A. Caenorhabditis elegans Aurora A kinase is required for the formation of spindle microtubules in female meiosis. Mol Biol Cell. 2015;26(23):4187–96. doi: 10.1091/mbc.E15-05-0258 26378257

62. Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974;77(1):71–94. 4366476

63. Paix A, Folkmann A, Rasoloson D, Seydoux G. High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR-Cas9 Ribonucleoprotein Complexes. Genetics. 2015;201(1):47–54. doi: 10.1534/genetics.115.179382 26187122

64. Kamath RS, Martinez-Campos M, Zipperlen P, Fraser AG, Ahringer J. Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome Biol. 2001;2(1):RESEARCH0002.

65. Fraser AG, Kamath RS, Zipperlen P, Martinez-Campos M, Sohrmann M, Ahringer J. Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature. 2000;408(6810):325–30. doi: 10.1038/35042517 11099033

66. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676–82. doi: 10.1038/nmeth.2019 22743772

67. Barry DJ, Durkin CH, Abella JV, Way M. Open source software for quantification of cell migration, protrusions, and fluorescence intensities. J Cell Biol. 2015;209(1):163–80. doi: 10.1083/jcb.201501081 25847537

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2020 Číslo 10

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