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Identifying Synaptic Proteins by In Vivo BioID from Mouse Brain

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Proximity Labeling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2008))

Abstract

Two anatomically and functionally distinct types of synapses are present in the central nervous system, excitatory synapses, and inhibitory synapses. Purification and analysis of the protein complex at the excitatory postsynapses have led to fundamental insights into neurobiology. In contrast, the biochemical purification and analysis of the inhibitory postsynaptic density have been largely intractable. The recently developed method called BioID employs the biotin ligase mutant, BirA*, fused to a bait protein to label and capture proximal proteins. We adapted the BioID approach to enable in vivo BioID, or iBioID of inhibitory synaptic complexes in the mouse brain. This protocol describes the iBioID method to allow synaptic bait proteins to target synaptic complexes, label, and purify biotinylated proteins from the mouse brain. This technique can be easily adapted to target other substructures in vivo that have been difficult to purify and analyze in the past.

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References

  1. Grant SG (2012) Synaptopathies: diseases of the synaptome. Curr Opin Neurobiol 22(3):522–529. https://doi.org/10.1016/j.conb.2012.02.002

    Article  CAS  Google Scholar 

  2. Volk L, Chiu SL, Sharma K, Huganir RL (2015) Glutamate synapses in human cognitive disorders. Annu Rev Neurosci 38:127–149. https://doi.org/10.1146/annurev-neuro-071714-033821

    Article  CAS  Google Scholar 

  3. Krueger-Burg D, Papadopoulos T, Brose N (2017) Organizers of inhibitory synapses come of age. Curr Opin Neurobiol 45:66–77. https://doi.org/10.1016/j.conb.2017.04.003

    Article  CAS  Google Scholar 

  4. Fritschy JM, Panzanelli P, Tyagarajan SK (2012) Molecular and functional heterogeneity of GABAergic synapses. Cell Mol Life Sci 69(15):2485–2499. https://doi.org/10.1007/s00018-012-0926-4

    Article  CAS  Google Scholar 

  5. Uezu A, Kanak DJ, Bradshaw TW, Soderblom EJ, Catavero CM, Burette AC, Weinberg RJ, Soderling SH (2016) Identification of an elaborate complex mediating postsynaptic inhibition. Science (New York, NY) 353(6304):1123–1129. https://doi.org/10.1126/science.aag0821

    Article  CAS  Google Scholar 

  6. Heller EA, Zhang W, Selimi F, Earnheart JC, Ślimak MA, Santos-Torres J, Ibañez-Tallon I, Aoki C, Chait BT, Heintz N (2012) The biochemical anatomy of cortical inhibitory synapses. PLoS One 7(6):e39572. https://doi.org/10.1371/journal.pone.0039572

    Article  CAS  Google Scholar 

  7. Kang Y, Ge Y, Cassidy RM, Lam V, Luo L, Moon K-M, Lewis R, Molday RS, Wong ROL, Foster LJ, Craig AM (2014) A combined transgenic proteomic analysis and regulated trafficking of neuroligin-2. J Biol Chem 289(42):29350–29364. https://doi.org/10.1074/jbc.M114.549279

    Article  CAS  Google Scholar 

  8. Nakamura Y, Morrow DH, Modgil A, Huyghe D, Deeb TZ, Lumb MJ, Davies PA, Moss SJ (2016) Proteomic characterization of inhibitory synapses using a novel pHluorin-tagged GABAAR α2 subunit knock-in mouse. J Biol Chem 291(23):12394–12407. https://doi.org/10.1074/jbc.M116.724443

    Article  CAS  Google Scholar 

  9. Ge Y, Kang Y, Cassidy RM, Moon KM, Lewis R, Wong ROL, Foster LJ, Craig AM (2018) Clptm1 limits forward trafficking of GABAA receptors to scale inhibitory synaptic strength. Neuron 97(3):596–610 e598. https://doi.org/10.1016/j.neuron.2017.12.038

    Article  CAS  Google Scholar 

  10. Davenport EC, Pendolino V, Kontou G, McGee TP, Sheehan DF, López-Doménech G, Farrant M, Kittler JT (2017) An essential role for the Tetraspanin LHFPL4 in the cell-type-specific targeting and clustering of synaptic GABA a receptors. Cell Rep 21(1):70–83

    Article  CAS  Google Scholar 

  11. Tokiwa Yamasaki, Erika Hoyos-Ramirez, James S. Martenson, Megumi Morimoto-Tomita, Susumu Tomita (2017) GARLH Family Proteins Stabilize GABA A Receptors at Synapses. Neuron 93(5):1138–1152.e6

    Article  Google Scholar 

  12. Min Wu, Hong-Lei Tian, Xiaobo Liu, John Ho Chun Lai, Shengwang Du, Jun Xia (2018) Impairment of Inhibitory Synapse Formation and Motor Behavior in Mice Lacking the NL2 Binding Partner LHFPL4/GARLH4. Cell Rep 23(6):1691–1705

    Article  CAS  Google Scholar 

  13. Roux KJ, Kim DI, Raida M, Burke B (2012) A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J Cell Biol 196(6):801–810. https://doi.org/10.1083/jcb.201112098

    Article  CAS  Google Scholar 

  14. Spector R, Mock D (1987) Biotin transport through the blood-brain barrier. J Neurochem 48(2):400–404

    Article  CAS  Google Scholar 

  15. Loh KH, Stawski PS, Draycott AS, Udeshi ND, Lehrman EK, Wilton DK, Svinkina T, Deerinck TJ, Ellisman MH, Stevens B, Carr SA, Ting AY (2016) Proteomic analysis of unbounded cellular compartments: synaptic clefts. Cell 166(5):1295–1307.e1221. https://doi.org/10.1016/j.cell.2016.07.041

    Article  CAS  Google Scholar 

  16. Tyagarajan SK, Fritschy JM (2014) Gephyrin: a master regulator of neuronal function? Nat Rev Neurosci 15(3):141–156. https://doi.org/10.1038/nrn3670

    Article  CAS  Google Scholar 

  17. Tretter V, Mukherjee J, Maric HM, Schindelin H, Sieghart W, Moss SJ (2012) Gephyrin, the enigmatic organizer at GABAergic synapses. Front Cell Neurosci 6:23. https://doi.org/10.3389/fncel.2012.00023

    Article  CAS  Google Scholar 

  18. Kim DI, Birendra KC, Zhu W, Motamedchaboki K, Doye V, Roux KJ (2014) Probing nuclear pore complex architecture with proximity-dependent biotinylation. Proc Natl Acad Sci U S A 111(24):E2453–E2461. https://doi.org/10.1073/pnas.1406459111

    Article  CAS  Google Scholar 

  19. Poulopoulos A, Aramuni G, Meyer G, Soykan T, Hoon M, Papadopoulos T, Zhang M, Paarmann I, Fuchs C, Harvey K, Jedlicka P, Schwarzacher SW, Betz H, Harvey RJ, Brose N, Zhang W, Varoqueaux F (2009) Neuroligin 2 drives postsynaptic assembly at perisomatic inhibitory synapses through gephyrin and collybistin. Neuron 63(5):628–642. https://doi.org/10.1016/j.neuron.2009.08.023

    Article  CAS  Google Scholar 

  20. Kins S, Betz H, Kirsch J (2000) Collybistin, a newly identified brain-specific GEF, induces submembrane clustering of gephyrin. Nat Neurosci 3(1):22–29. https://doi.org/10.1038/71096

    Article  CAS  Google Scholar 

  21. Kim DI, Jensen SC, Noble KA, Kc B, Roux KH, Motamedchaboki K, Roux KJ (2016) An improved smaller biotin ligase for BioID proximity labeling. Mol Biol Cell 27(8):1188–1196. https://doi.org/10.1091/mbc.E15-12-0844

    Article  CAS  Google Scholar 

  22. Branon TC, Bosch JA, Sanchez AD, Udeshi ND, Svinkina T, Carr SA, Feldman JL, Perrimon N, Ting AY (2017) Directed evolution of TurboID for efficient proximity labeling in living cells and organisms. bioRxiv. 196980. https://doi.org/10.1101/196980

  23. Ramanathan M, Majzoub K, Rao DS, Neela PH, Zarnegar BJ, Mondal S, Roth JG, Gai H, Kovalski JR, Siprashvili Z, Palmer TD, Carette JE, Khavari PA (2018) RNA-protein interaction detection in living cells. Nat Methods 15(3):207–212. https://doi.org/10.1038/nmeth.4601

    Article  CAS  Google Scholar 

  24. Daigle TL, Madisen L, Hage TA, Valley MT, Knoblich U, Larsen RS, Takeno MM, Huang L, Gu H, Larsen R, Mills M, Bosma-Moody A, Siverts LA, Walker M, Graybuck LT, Yao Z, Fong O, Garren E, Lenz G, Chavarha M, Pendergraft J, Harrington J, Hirokawa KE, Harris JA, McGraw M, Ollerenshaw DR, Smith K, Baker BA, Ting JT, Sunkin SM, Lecoq J, Lin MZ, Boyden ES, Murphy GJ, Costa ND, Waters J, Li L, Tasic B, Zeng H (2017) A suite of transgenic driver and reporter mouse lines with enhanced brain cell type targeting and functionality. bioRxiv. 224881. https://doi.org/10.1101/224881

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Acknowledgments

The authors would like to thank K. Sakurai and J. Takatoh for advice on AAV injection and C.M. Catavero and S. Zhao for technical assists of AAV production. We thank also T. Ohashi, M. Osawa, and T. Lechler for their suggestion to optimize the biotinylated protein purification step. iBioID method was further refined by the collaborative efforts of past and present Scott Soderling lab members. Credit of the protocol goes to Akiyoshi Uezu, and Scott Soderling. We are grateful to P. Devlin, J. Croucher, and E. Erata for feedback and suggestions during preparation of this chapter. This work is supported by NIH grants (MH104736, NS039444).

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Authors and Affiliations

  1. The Department of Cell Biology, Duke University Medical School, Durham, NC, USA

    Akiyoshi Uezu

  2. The Department of Cell Biology and Neurobiology, Duke University Medical School, Durham, NC, USA

    Scott Soderling

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  1. Akiyoshi Uezu
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  2. Scott Soderling
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Correspondence to Akiyoshi Uezu .

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Editors and Affiliations

  1. Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany

    Murat Sunbul  & Andres Jäschke  & 

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Uezu, A., Soderling, S. (2019). Identifying Synaptic Proteins by In Vivo BioID from Mouse Brain. In: Sunbul, M., Jäschke, A. (eds) Proximity Labeling. Methods in Molecular Biology, vol 2008. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9537-0_9

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  • DOI: https://doi.org/10.1007/978-1-4939-9537-0_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9536-3

  • Online ISBN: 978-1-4939-9537-0

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