Abstract
Protein–protein interactions (PPIs) play a central role in all cellular processes. The discovery of green fluorescent protein (GFP) and split varieties, which are functionally reconstituted by complementation, led to the development of the bimolecular fluorescence complementation (BiFC) assay for the investigation of PPI in vivo. BiFC became a popular tool, as it is a convenient and quick technology to directly visualize PPI in a wide variety of living cells. In combination with the transparency of the early zebrafish embryo, it also permits detection of PPI in the context of an entire living organism, which performs all spatial and temporal regulations missing in in vitro systems like tissue culture. However, the application of BiFC in some research areas including the study of zebrafish is limited due to the lack of efficient and convenient BiFC expression vectors. Here, we describe the engineering of a novel set of Gateway®-adapted BiFC destination vectors to investigate PPI with all possible permutations for BiFC experiments. Moreover, we demonstrate the versatility of these destination vectors by confirming the interaction between zebrafish Bucky ball and RNA helicase Vasa in living embryos.
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References
Filonov GS, Verkhusha VV (2013) A near-infrared bifc reporter for in vivo imaging of protein-protein interactions. Chem Biol 20:1078–1086. https://doi.org/10.1016/j.chembiol.2013.06.009
Kerppola TK (2006) Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells. Nat Protoc 1:1278–1286. https://doi.org/10.1038/nprot.2006.201
Kerppola TK (2008) Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells. Annu Rev Biophys 37:465–487. https://doi.org/10.1146/annurev.biophys.37.032807.125842.BIMOLECULAR
Kodama Y, Hu CD (2012) Bimolecular fluorescence complementation (BiFC): a 5-year update and future perspectives. BioTechniques 53:285–298. https://doi.org/10.2144/000113943
Ghosh I, Hamilton AD, Regan L et al (2000) Antiparallel leucine zipper-directed protein reassembly: application to the green fluorescent protein. J Am Chem Soc 122(23):5658–5659
Zhong S, Lin Z, Fray RG, Grierson D (2008) Improved plant transformation vectors for fluorescent protein tagging. Transgenic Res 17:985–989. https://doi.org/10.1007/s11248-008-9199-y
Free RB, Hazelwood LA, Sibley DR (2009) Identifying novel protein-protein interactions using co-IP and mass spectrometry. Curr Protoc Neurosci 2:1–19. https://doi.org/10.1002/0471142301.ns0528s46.Identifying
Kamigaki A, Nito K, Hikino K et al (2016) Gateway vectors for simultaneous detection of multiple protein-protein interactions in plant cells using bimolecular fluorescence complementation. PLoS One 11:1–15. https://doi.org/10.1371/journal.pone.0160717
Zhang XE, Cui Z, Wang D (2016) Sensing of biomolecular interactions using fluorescence complementing systems in living cells. Biosens Bioelectron 76:243–250. https://doi.org/10.1016/j.bios.2015.07.069
Cieri D, Vicario M, Giacomello M et al (2018) SPLICS: A split green fluorescent protein-based contact site sensor for narrow and wide heterotypic organelle juxtaposition. Cell Death Differ 25:1131–1145. https://doi.org/10.1038/s41418-017-0033-z
Feng S, Varshney A, Villa DC et al (2018) Bright split red fluorescent proteins with enhanced complementation efficiency for the tagging of endogenous proteins and visualization of synapses. bioRxiv 2018:454041. https://doi.org/10.1101/454041
Han Y, Wang S, Zhang Z et al (2014) In vivo imaging of protein-protein and RNA-protein interactions using novel far-red fluorescence complementation systems. Nucleic Acids Res 2014:42. https://doi.org/10.1093/nar/gku408
Bischof J, Duffraisse M, Furger E et al (2018) Generation of a versatile bifc orfeome library for analyzing protein–protein interactions in live drosophila. elife 7:1–24. https://doi.org/10.7554/eLife.38853
Summary M (2018) Kusabira-Orange protein:64. https://doi.org/10.4155/btn-2017-0121
Verhoef LGGC, Wade M (2017) Visualization of protein interactions in living cells using bimolecular luminescence complementation (BiLC). Curr Protoc Protein Sci 90:30.5.1–30.5.14. https://doi.org/10.1002/cpps.42
Miles LB, Verkade H (2014) TA-cloning vectors for rapid and cheap cloning of zebrafish transgenesis constructs. Zebrafish 11:281–282. https://doi.org/10.1089/zeb.2013.0954
Villefranc JA, Amigo J, Lawson ND (2007) Gateway compatible vectors for analysis of gene function in the zebrafish. Dev Dyn 236:3077–3087. https://doi.org/10.1002/dvdy.21354
Kwan KM, Fujimoto E, Grabher C et al (2007) The Tol2kit: a multisite gateway-based construction Kit for Tol2 transposon transgenesis constructs. Dev Dyn 236:3088–3099. https://doi.org/10.1002/dvdy.21343
Krishnakumar P, Riemer S, Perera R et al (2018) Functional equivalence of germ plasm organizers. PLoS Genet 14:1–29. https://doi.org/10.1371/journal.pgen.1007696
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Perera, R.P., Dosch, R. (2021). In Vivo Imaging of Protein Interactions in the Germplasm with Bimolecular Fluorescent Complementation. In: Dosch, R. (eds) Germline Development in the Zebrafish. Methods in Molecular Biology, vol 2218. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0970-5_24
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DOI: https://doi.org/10.1007/978-1-0716-0970-5_24
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