Abstract
The ability to identify protein interactions is key to elucidating the molecular mechanisms of cellular processes, including mitosis and cell cycle regulation. Drosophila melanogaster, as a model system, provides powerful tools to study cell division using genetics, microscopy, and RNAi. Drosophila early embryos are highly enriched in mitotic protein complexes as their nuclei undergo 13 rounds of rapid, synchronous mitotic nuclear divisions in a syncytium during the first 2 h of development. Here, we describe simple methods for the affinity purification of protein complexes from transgenic fly embryos via protein A- and green fluorescent protein-tags fused to bait proteins of interest. This in vivo proteomics approach has allowed the identification of several known and novel mitotic protein interactions using mass spectrometry, and it expands the use of the Drosophila model in modern molecular biology.
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References
Dunham WH, Mullin M, Gingras AC (2012) Affinity-purification coupled to mass spectrometry: basic principles and strategies. Proteomics 12(10):1576–1590
Walther TC, Mann M (2010) Mass spectrometry-based proteomics in cell biology. J Cell Biol 190(4):491–500
Archambault V (2005) Cell cycle: proteomics gives it a spin. Expert Rev Proteomics 2(4):615–625
D’Avino PP, Archambault V, Przewloka MR, Zhang W, Laue ED, Glover DM (2009) Isolation of protein complexes involved in mitosis and cytokinesis from Drosophila cultured cells. Methods Mol Biol 545:99–112
Przewloka MR, Zhang W, Costa P, Archambault V, D’Avino PP, Lilley KS, Laue ED, McAinsh AD, Glover DM (2007) Molecular analysis of core kinetochore composition and assembly in Drosophila melanogaster. PLoS One 2:e478
Archambault V, D’Avino PP, Deery MJ, Lilley KS, Glover DM (2008) Sequestration of Polo kinase to microtubules by phosphopriming-independent binding to Map205 is relieved by phosphorylation at a CDK site in mitosis. Genes Dev 22(19):2707–2720
Montembault E, Zhang W, Przewloka MR, Archambault V, Sevin EW, Laue ED, Glover DM, D’Avino PP (2010) Nessun Dorma, a novel centralspindlin partner, is required for cytokinesis in Drosophila spermatocytes. J Cell Biol 191(7):1351–1365
Foe VE, Odell GM, Edgar BA (1993) Mitosis and morphogenesis in the Drosophila embryo: point and counterpoint. In: Bate M, Martinez Arias A (eds) The development of Drosophila melanogaster, vol 1. Cold Spring Harbor Laboratory Press, New York
Lipinszki Z, Klement E, Hunyadi-Gulyas E, Medzihradszky KF, Markus R, Pal M, Deak P, Udvardy A (2013) A novel interplay between the ubiquitin-proteasome system and serine proteases during Drosophila development. Biochem J 454(3):571–583
Ashburner M, Golic KG, Hawley RS (2005) Drosophila, a laboratory handbook, 2nd edn. Cold Spring Harbor Laboratory Press, New York
Duffy JB (2002) GAL4 system in Drosophila: a fly geneticist’s Swiss army knife. Genesis 34(1–2):1–15
Rorth P (1998) Gal4 in the Drosophila female germline. Mech Dev 78(1–2):113–118
Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118(2):401–415
Kunert N, Brehm A (2008) Mass production of Drosophila embryos and chromatographic purification of native protein complexes. Methods Mol Biol 420:359–371
Neumuller RA, Wirtz-Peitz F, Lee S, Kwon Y, Buckner M, Hoskins RA, Venken KJ, Bellen HJ, Mohr SE, Perrimon N (2012) Stringent analysis of gene function and protein-protein interactions using fluorescently tagged genes. Genetics 190(3):931–940
Moutinho-Santos T, Sampaio P, Amorim I, Costa M, Sunkel CE (1999) In vivo localisation of the mitotic POLO kinase shows a highly dynamic association with the mitotic apparatus during early embryogenesis in Drosophila. Biol Cell 91(8):585–596
Buffin E, Lefebvre C, Huang J, Gagou ME, Karess RE (2005) Recruitment of Mad2 to the kinetochore requires the Rod/Zw10 complex. Curr Biol 15(9):856–861
Acknowledgements
This work was supported by grants from the Canadian Institutes of Health Research (CIHR) and the Natural Sciences and Engineering Research Council of Canada (to V.A.) and by Cancer Research UK, Medical Research Council, and Biotechnology and Biological Sciences Research Council grants (to D.M.G.). V.A. holds a New Investigator Award from the CIHR. IRIC is supported in part by the Canada Foundation for Innovation, and the FRQS. Z.L. holds the Long-term Fellowship of the Federation of European Biochemical Societies (FEBS). Some of the destination vectors for the Gateway cloning were obtained from the Drosophila Genomics Research Centre. We are grateful to Janusz Debski and Michal Dadlez (MS LAB, Institute of Biochemistry and Biophysics PAS, Warsaw, Poland) and Pierre Thibault and Éric Bonneil (IRIC, Université de Montréal) for mass spectrometric analysis.
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Lipinszki, Z. et al. (2014). Affinity Purification of Protein Complexes from Drosophila Embryos in Cell Cycle Studies. In: Noguchi, E., Gadaleta, M. (eds) Cell Cycle Control. Methods in Molecular Biology, vol 1170. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0888-2_33
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DOI: https://doi.org/10.1007/978-1-4939-0888-2_33
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