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