SynGAP forms biocondensates at sub-micromolar concentrations and recruits PSD95 and receptor oligomers, functioning as a key initiator of PSD formation

Creators: Acharya, Saahil¹; Tsunoyama, Taka A.¹; Hoffmann, Christian²; Aguilar, Perez Gerard²; Meshcheryakova, Irina¹; Nakamura-Norimoto, Aya¹; Mastro, Tara³; Walkup, Ward G., IV³; Fujiwara, Takahiro K.⁴; Kennedy, Mary B.³; Milovanovic, Dragomir²; Kusumi, Akihiro^{1, 4}

1. Okinawa Institute of Science and Technology
2. German Center for Neurodegenerative Diseases
3. California Institute of Technology
4. Kyoto University

Abstract

A key issue in neuronal circuit regulation is how synapse formation is initiated. Synapse formation could start when one or more synaptic scaffold proteins that can initiate synapse formation reach certain threshold concentrations in the dendritic shaft, which might lead to their oligomerization or even liquid-liquid phase separation (LLPS). By combining in vitro reconstitution of purified proteins with live-cell single-molecule and confocal imaging, we demonstrated that SynGAP alone forms assemblies of nanoscale clusters containing several to several tens of molecules at 10-nM order concentrations and micron-scale LLPS hydrogel-like condensates at submicromolar concentrations. The trimers of SynGAP’s intrinsically disordered region (IDR) induced by its coiled-coil domain are responsible for SynGAP condensation. CaMKII-mediated phosphorylation moderately suppresses SynGAP condensation, and also increases condensate liquidity. While PSD95 fails to form assemblies under these conditions, it is recruited to SynGAP condensates by specifically binding to the PDZ-binding motif of SynGAP. SynGAP[PSD95] condensates selectively immobilize postsynaptic transmembrane proteins, Neuroligin1 and AMPAR-TARP2 complexes, in a manner dependent on their oligomerization state, indicating cooperative recruitment dynamics among SynGAP, PSD95, and transmembrane components, which might mimic initial PSD assembly. These findings suggest that SynGAP may act as a primary nucleator of postsynaptic density assembly, challenging the PSD95-centered models.

Copyright and License

The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.

Supplemental Material

Supplementary Tables[supplements/649955_file04.docx]

Acknowledgement

We thank Dr. Yuri Nemoto and Ms. Hiroko Hijikata for their kind instructions for the primary culture of rat hippocampal neurons and Profs. Ikuko Koyama-Honda and Noboru Mizushima of the University of Tokyo for their kind help and instruction about the methods for producing viral vectors and viruses and cell infection. We thank Prof. Peter Scheiffele of University of Basel, Prof. Shigeo Okabe of the University of Tokyo, Prof. Tomoo Hirano of Kyoto University, and Prof. Michiyuki Matsuda of Kyoto University for their kind gifts of Nlg1 cDNA, PSD95 cDNA, pCAGplay vector, and pBpuro and pCMV-mPbase plasmids, respectively. We also thank the members of the Kusumi laboratory, Milovanovic laboratory, and Kennedy laboratory for helpful discussions.

Funding

This work was supported in part by Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (Kiban A to A.K. [21H04772], Kiban S to A.K. [16H06386], Kiban B to T.K.F. [16H04775], Kiban C to R.S.K. [17K07333], Wakate to T.A.T. [21K15058], and Challenging Exploratory Research to T.K.F. [18K19001] and A.K. [22K19334]), and a Japan Science and Technology grant ACT-X to T.A.T. (JPMJAX211B). WPI-iCeMS of Kyoto University is supported by the World Premiere Research Center Initiative (WPI) of MEXT.

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