Fluorescence fluctuation analysis reveals PpV dependent Cdc25 protein dynamics in living embryos

Autoři: Boyang Liu aff001;  Ingo Gregor aff003;  H.-Arno Müller aff004;  Jörg Großhans aff001
Působiště autorů: Fachbereich Biologie (FB17), Philipps-Universität Marburg, Marburg, Germany aff001;  Institut für Entwicklungsbiochemie, Universitätsmedizin, Georg-August-Universität Göttingen, Göttingen, Germany aff002;  Drittes Physikalisches Institut, Georg-August-Universität Göttingen, Göttingen, Germany aff003;  Fachgebiet Entwicklungsgenetik, Institut für Biologie, Universität Kassel, Kassel, Germany aff004
Vyšlo v časopise: Fluorescence fluctuation analysis reveals PpV dependent Cdc25 protein dynamics in living embryos. PLoS Genet 16(4): e32767. doi:10.1371/journal.pgen.1008735
Kategorie: Research Article
doi: 10.1371/journal.pgen.1008735


The protein phosphatase Cdc25 is a key regulator of the cell cycle by activating Cdk-cyclin complexes. Cdc25 is regulated by its expression levels and post-translational mechanisms. In early Drosophila embryogenesis, Cdc25/Twine drives the fast and synchronous nuclear cycles. A pause in the cell cycle and the remodeling to a more generic cell cycle mode with a gap phase are determined by Twine inactivation and destruction in early interphase 14, in response to zygotic genome activation. Although the pseudokinase Tribbles contributes to the timely degradation of Twine, Twine levels are controlled by additional yet unknown post-translational mechanisms. Here, we apply a non-invasive method based on fluorescence fluctuation analysis (FFA) to record the absolute concentration profiles of Twine with minute-scale resolution in single living embryos. Employing this assay, we found that Protein phosphatase V (PpV), the homologue of the catalytic subunit of human PP6, ensures appropriately low Twine protein levels at the onset of interphase 14. PpV controls directly or indirectly the phosphorylation of Twine at multiple serine and threonine residues as revealed by phosphosite mapping. Mutational analysis confirmed that these sites are involved in control of Twine protein dynamics, and cell cycle remodeling is delayed in a fraction of the phosphosite mutant embryos. Our data reveal a novel mechanism for control of Twine protein levels and their significance for embryonic cell cycle remodeling.

Klíčová slova:

Cell cycle and cell division – Drosophila melanogaster – Embryos – Invertebrate genomics – Mitosis – Phosphatases – Phosphorylation – Fluctuation analysis


1. Krek W, Nigg EA. Differential phosphorylation of vertebrate p34cdc2 kinase at the G1/S and G2/M transitions of the cell cycle: identification of major phosphorylation sites. EMBO J. 1991;10(2):305–16. 1846803

2. Parker LL, Piwnica-Worms H. Inactivation of the p34cdc2-cyclin B complex by the human WEE1 tyrosine kinase. Science. 1992;257(5078):1955–7. doi: 10.1126/science.1384126 1384126

3. Lucena R, Alcaide-Gavilan M, Anastasia SD, Kellogg DR. Wee1 and Cdc25 are controlled by conserved PP2A-dependent mechanisms in fission yeast. Cell Cycle. 2017;16(5):428–35. doi: 10.1080/15384101.2017.1281476 28103117

4. Perry JA, Kornbluth S. Cdc25 and Wee1: analogous opposites? Cell Div. 2007;2:12. doi: 10.1186/1747-1028-2-12 17480229

5. Farrell JA, O'Farrell PH. From egg to gastrula: how the cell cycle is remodeled during the Drosophila mid-blastula transition. Annu Rev Genet. 2014;48:269–94. doi: 10.1146/annurev-genet-111212-133531 25195504

6. Liu B, Grosshans J. Link of Zygotic Genome Activation and Cell Cycle Control. Methods Mol Biol. 2017;1605:11–30. doi: 10.1007/978-1-4939-6988-3_2 28456955

7. Liu B, Winkler F, Herde M, Witte CP, Grosshans J. A Link between Deoxyribonucleotide Metabolites and Embryonic Cell-Cycle Control. Curr Biol. 2019;29(7):1187–92 e3. doi: 10.1016/j.cub.2019.02.021 30880011

8. Blythe SA, Wieschaus EF. Zygotic genome activation triggers the DNA replication checkpoint at the midblastula transition. Cell. 2015;160(6):1169–81. doi: 10.1016/j.cell.2015.01.050 25748651

9. Djabrayan NJ, Smits CM, Krajnc M, Stern T, Yamada S, Lemon WC, et al. Metabolic Regulation of Developmental Cell Cycles and Zygotic Transcription. Curr Biol. 2019;29(7):1193–8 e5. doi: 10.1016/j.cub.2019.02.028 30880009

10. Vastenhouw NL, Cao WX, Lipshitz HD. The maternal-to-zygotic transition revisited. Development. 2019;146(11).

11. Liu B, Grosshans J. The role of dNTP metabolites in control of the embryonic cell cycle. Cell Cycle. 2019;18(21):2817–27. doi: 10.1080/15384101.2019.1665948 31544596

12. Sung HW, Spangenberg S, Vogt N, Grosshans J. Number of nuclear divisions in the Drosophila blastoderm controlled by onset of zygotic transcription. Curr Biol. 2013;23(2):133–8. doi: 10.1016/j.cub.2012.12.013 23290555

13. Grosshans J, Muller HA, Wieschaus E. Control of cleavage cycles in Drosophila embryos by fruhstart. Dev Cell. 2003;5(2):285–94. doi: 10.1016/s1534-5807(03)00208-9 12919679

14. Grosshans J, Wieschaus E. A genetic link between morphogenesis and cell division during formation of the ventral furrow in Drosophila. Cell. 2000;101(5):523–31. doi: 10.1016/s0092-8674(00)80862-4 10850494

15. Gawlinski P, Nikolay R, Goursot C, Lawo S, Chaurasia B, Herz HM, et al. The Drosophila mitotic inhibitor Fruhstart specifically binds to the hydrophobic patch of cyclins. EMBO Rep. 2007;8(5):490–6. doi: 10.1038/sj.embor.7400948 17431409

16. Alphey L, Jimenez J, White-Cooper H, Dawson I, Nurse P, Glover DM. twine, a cdc25 homolog that functions in the male and female germline of Drosophila. Cell. 1992;69(6):977–88. doi: 10.1016/0092-8674(92)90616-k 1606618

17. Courtot C, Fankhauser C, Simanis V, Lehner CF. The Drosophila cdc25 homolog twine is required for meiosis. Development. 1992;116(2):405–16. 1286615

18. Edgar BA, Datar SA. Zygotic degradation of two maternal Cdc25 mRNAs terminates Drosophila's early cell cycle program. Genes Dev. 1996;10(15):1966–77. doi: 10.1101/gad.10.15.1966 8756353

19. Farrell JA, O'Farrell PH. Mechanism and regulation of Cdc25/Twine protein destruction in embryonic cell-cycle remodeling. Curr Biol. 2013;23(2):118–26. doi: 10.1016/j.cub.2012.11.036 23290551

20. Farrell JA, Shermoen AW, Yuan K, O'Farrell PH. Embryonic onset of late replication requires Cdc25 down-regulation. Genes Dev. 2012;26(7):714–25. doi: 10.1101/gad.186429.111 22431511

21. Di Talia S, She R, Blythe SA, Lu X, Zhang QF, Wieschaus EF. Posttranslational control of Cdc25 degradation terminates Drosophila's early cell-cycle program. Curr Biol. 2013;23(2):127–32. doi: 10.1016/j.cub.2012.11.029 23290553

22. Mata J, Curado S, Ephrussi A, Rorth P. Tribbles coordinates mitosis and morphogenesis in Drosophila by regulating string/CDC25 proteolysis. Cell. 2000;101(5):511–22. doi: 10.1016/s0092-8674(00)80861-2 10850493

23. Seher TC, Leptin M. Tribbles, a cell-cycle brake that coordinates proliferation and morphogenesis during Drosophila gastrulation. Curr Biol. 2000;10(11):623–9. doi: 10.1016/s0960-9822(00)00502-9 10837248

24. Frazer C, Young PG. Phosphorylation Mediated Regulation of Cdc25 Activity, Localization and Stability In Protein Phosphorylation in Human Health. In: Huang C, editor. InTech. Rijeka, Croatia: InTech; 2009. p. 395–436.

25. Mann DJ, Dombradi V, Cohen PT. Drosophila protein phosphatase V functionally complements a SIT4 mutant in Saccharomyces cerevisiae and its amino-terminal region can confer this complementation to a heterologous phosphatase catalytic domain. EMBO J. 1993;12(12):4833–42. 8223492

26. Bastians H, Ponstingl H. The novel human protein serine/threonine phosphatase 6 is a functional homologue of budding yeast Sit4p and fission yeast ppe1, which are involved in cell cycle regulation. J Cell Sci. 1996;109 (Pt 12):2865–74.

27. Ohama T. The multiple functions of protein phosphatase 6. Biochim Biophys Acta Mol Cell Res. 2019;1866(1):74–82. doi: 10.1016/j.bbamcr.2018.07.015 30036567

28. Liu B, Sung HW, Grosshans J. Multiple Functions of the Essential Gene PpV in Drosophila Early Development. G3 (Bethesda). 2019;9(11):3583–93.

29. Yu SR, Burkhardt M, Nowak M, Ries J, Petrasek Z, Scholpp S, et al. Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules. Nature. 2009;461(7263):533–6. doi: 10.1038/nature08391 19741606

30. Ries J, Yu SR, Burkhardt M, Brand M, Schwille P. Modular scanning FCS quantifies receptor-ligand interactions in living multicellular organisms. Nat Methods. 2009;6(9):643–5. doi: 10.1038/nmeth.1355 19648917

31. Abu-Arish A, Porcher A, Czerwonka A, Dostatni N, Fradin C. High mobility of bicoid captured by fluorescence correlation spectroscopy: implication for the rapid establishment of its gradient. Biophys J. 2010;99(4):L33–5. doi: 10.1016/j.bpj.2010.05.031 20712981

32. Bhattacharya D, Talwar S, Mazumder A, Shivashankar GV. Spatio-temporal plasticity in chromatin organization in mouse cell differentiation and during Drosophila embryogenesis. Biophys J. 2009;96(9):3832–9. doi: 10.1016/j.bpj.2008.11.075 19413989

33. Chen Y, Muller JD, Ruan Q, Gratton E. Molecular brightness characterization of EGFP in vivo by fluorescence fluctuation spectroscopy. Biophys J. 2002;82(1 Pt 1):133–44.

34. Digman MA, Dalal R, Horwitz AF, Gratton E. Mapping the number of molecules and brightness in the laser scanning microscope. Biophys J. 2008;94(6):2320–32. doi: 10.1529/biophysj.107.114645 18096627

35. Buschmann V, Kramer B, Koberlink F. Quantitative FCS: Determination of the Confocal Volume by FCS and Bead Scanning with MicroTime 200. PicoQuant. Berlin: Application Note; 2009. p. 8.

36. Mazzolini L, Broban A, Froment C, Burlet-Schiltz O, Besson A, Manenti S, et al. Phosphorylation of CDC25A on SER283 in late S/G2 by CDK/cyclin complexes accelerates mitotic entry. Cell Cycle. 2016;15(20):2742–52. doi: 10.1080/15384101.2016.1220455 27580187

37. Mailand N, Podtelejnikov AV, Groth A, Mann M, Bartek J, Lukas J. Regulation of G(2)/M events by Cdc25A through phosphorylation-dependent modulation of its stability. EMBO J. 2002;21(21):5911–20. doi: 10.1093/emboj/cdf567 12411508

38. Donzelli M, Squatrito M, Ganoth D, Hershko A, Pagano M, Draetta GF. Dual mode of degradation of Cdc25 A phosphatase. EMBO J. 2002;21(18):4875–84. doi: 10.1093/emboj/cdf491 12234927

39. Shimuta K, Nakajo N, Uto K, Hayano Y, Okazaki K, Sagata N. Chk1 is activated transiently and targets Cdc25A for degradation at the Xenopus midblastula transition. EMBO J. 2002;21(14):3694–703. doi: 10.1093/emboj/cdf357 12110582

40. Di Talia S, Wieschaus EF. Short-term integration of Cdc25 dynamics controls mitotic entry during Drosophila gastrulation. Dev Cell. 2012;22(4):763–74. doi: 10.1016/j.devcel.2012.01.019 22483720

41. Stumpff J, Duncan T, Homola E, Campbell SD, Su TT. Drosophila Wee1 kinase regulates Cdk1 and mitotic entry during embryogenesis. Curr Biol. 2004;14(23):2143–8. doi: 10.1016/j.cub.2004.11.050 15589158

42. Price D, Rabinovitch S, O'Farrell PH, Campbell SD. Drosophila wee1 has an essential role in the nuclear divisions of early embryogenesis. Genetics. 2000;155(1):159–66. 10790391

43. Deneke VE, Melbinger A, Vergassola M, Di Talia S. Waves of Cdk1 Activity in S Phase Synchronize the Cell Cycle in Drosophila Embryos. Dev Cell. 2016;38(4):399–412. doi: 10.1016/j.devcel.2016.07.023 27554859

44. Whitworth C. The Bloomington Drosophila Stock Center: Management, Maintenance, Distribution, and Research. In: Jarret RL, McCluskey K, editors. The Biological Resources of Model Organisms. London: Taylor & Francis Group; 2019. p. 145–62.

45. Gramates LS, Marygold SJ, Santos GD, Urbano JM, Antonazzo G, Matthews BB, et al. FlyBase at 25: looking to the future. Nucleic Acids Res. 2017;45(D1):D663–D71. doi: 10.1093/nar/gkw1016 27799470

46. Yan S, Acharya S, Groning S, Grosshans J. Slam protein dictates subcellular localization and translation of its own mRNA. PLoS Biol. 2017;15(12):e2003315. doi: 10.1371/journal.pbio.2003315 29206227

47. Wenzl C, Yan S, Laupsien P, Grosshans J. Localization of RhoGEF2 during Drosophila cellularization is developmentally controlled by Slam. Mech Dev. 2010;127(7–8):371–84. doi: 10.1016/j.mod.2010.01.001 20060902

48. Acharya S, Laupsien P, Wenzl C, Yan S, Grosshans J. Function and dynamics of slam in furrow formation in early Drosophila embryo. Dev Biol. 2014;386(2):371–84. doi: 10.1016/j.ydbio.2013.12.022 24368071

49. Winkler F, Kriebel M, Clever M, Groning S, Grosshans J. Essential Function of the Serine Hydroxymethyl Transferase (SHMT) Gene During Rapid Syncytial Cell Cycles in Drosophila. G3 (Bethesda). 2017;7(7):2305–14.

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PLOS Genetics

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