Identification of an elaborate complex mediating postsynaptic inhibition

Abstract

Inhibitory synapses dampen neuronal activity through postsynaptic hyperpolarization. The composition of the inhibitory postsynapse and the mechanistic basis of its regulation, however, remains poorly understood. We used an in vivo chemico-genetic proximity-labeling approach to discover inhibitory postsynaptic proteins. Quantitative mass spectrometry not only recapitulated known inhibitory postsynaptic proteins, but also revealed a large network of new proteins, many of which are either implicated in neurodevelopmental disorders or are of unknown function. CRISPR-depletion of one of these previously uncharacterized proteins, InSyn1, led to decreased postsynaptic inhibitory sites, reduced frequency of miniature inhibitory currents, and increased excitability in the hippocampus. Our findings uncover a rich and functionally diverse assemblage of previously unknown proteins that regulate postsynaptic inhibition and might contribute to developmental brain disorders.

BioID: A Screen for Protein‐Protein Interactions

“…BioID has been used in mouse tumor xenograft studies and via viral transduction in the mouse brain (Chan et al, 2014; Uezu et al, 2016). Aprotein-complementation-based split-BioID has been generated in which the ligase is only active when two proteins, each fused to a complementary fragment of the ligase, are brought together by an interaction (De Munter et al, 2017).…”

BioID as a Tool for Protein-Proximity Labeling in Living Cells

“…Since its development and initial application in 2012 (7), BioID has been cited and/or applied in over 200 PubMed-published articles and has been utilized in several unicellular organisms (see (12–15) for examples), mammalian cells (5), plants (16,17), and mice (18–20), including compartmental proteomics of a parasitic organism infecting mice (21). Novel or improved applications using BioID ligase have spurred numerous follow-up articles including a smaller version of BioID with improved sensitivity and localization (22), its use for identifying protein-RNA interactions (23), split-BioID studies (24,25), and faster versions of BioID (26).…”

GABA type a receptor trafficking and the architecture of synaptic inhibition

“…Yet, advancement of new and existing methods is promoting major inroads for our understanding of GABA A R signaling and trafficking. Recent proteomic strategies have vastly expanded the protein repertoire of GABAergic synapses to nearly 200 proteins by targeted purification of GABA A Rs, NL2, or gephyrin (collybistin and InSyn1 to a lesser extent) and their respective interactomes (Kang et al, ; Loh et al, ; Nakamura et al, ; Uezu et al, ). Microscopy‐based approaches encompass the fastest growing means of discovery in vitro and in vivo , including optogenetically controlled GABA A Rs (Lin et al, ), two‐photon based GABA photolysis (Oh et al, ), single‐particle‐tracking of receptor diffusion (Petrini and Barberis, ), quantitative super‐resolution imaging of gephyrin (Pennacchietti et al, ) and gephyrin recombinant antibody‐like proteins (Gross et al, ) or fluorescent super‐binding peptides (Maric et al, ).…”