Biomolecular condensate assembly of nArgBP2 tunes its functionality to manifest the structural plasticity of dendritic spines

Abstract

AbstractnArgBP2, a candidate gene for intellectual disability, is a postsynaptic protein critical for dendritic spine development and morphogenesis, and its knockdown (KD) in developing neurons severely impairs spine-bearing excitatory synapse formation. Surprisingly, nArgBP2 KD in mature neurons did not cause morphological defects in the existing spines at rest, raising questions of how it functions in mature neurons. We found that unlike its inaction at rest, nArgBP2 KD completely inhibited the enlargement of dendritic spines during chemically induced long-term potentiation (cLTP) in mature neurons. We further found that nArgBP2 forms condensates in dendritic spines and that these condensates are dispersed by cLTP, which spatiotemporally coincides with spine head enlargement. Condensates with CaMKII phosphorylation-deficient mutant or CaMKII inhibition are neither dispersed nor accompanied by spine enlargement during cLTP. We found that nArgBP2 condensates in spines exhibited liquid-like properties, and in heterologous and in vitro expression systems, nArgBP2 undergoes liquid-liquid phase separation via multivalent intermolecular interactions between SH3 domains and proline-rich domains. It also forms coacervates with CaMKIIα, which is rapidly dissembled by calcium/CaMKIIα-dependent phosphorylation. We further showed that the interaction between nArgBP2 and WAVE1 competes with nArgBP2 phase separation and that blocking the nArgBP2-WAVE1 interaction prevents spine enlargement during cLTP. Together, our results suggest that nArgBP2 at rest is confined to the condensates but is released by CaMKIIα-mediated phosphorylation during synaptic plasticity, which regulates its timely interaction with WAVE1 to induce spine head enlargement in mature neurons.

nArgBP2 together with GKAP and SHANK3 forms a dynamic layered structure

“…This segregation process ultimately gives rise to the formation of a nanodomain-like structure within a single protein condensate ( Hosokawa et al, 2021 ). We also found that nArgBP2 was co-assembled into condensates with CaMKIIα in living COS7 cells and nArgBP2 condensates are regulated by CaMKIIα-mediated phosphorylation ( Cho et al, 2023 ).…”

“…We recently showed that nArgBP2 undergoes phase separation in vitro and in living cells ( Cho et al, 2023 ). PSD proteins including GKAP and SHANK also undergo LLPS in vitro ( Zeng et al, 2018 ), and recent studies suggest that LLPS is a key mechanism for the subcellular organization of post-synaptic assembly ( Chen et al, 2020 ).…”

“…EGFP – nArgBP2 959–1196 was generated as previously described ( Cho et al, 2023 ). Plasmids encoding HA-SHANK3 and GKAP-Myc were kind gifts from Prof. Jae-Yong Park (Korea University, Seoul, South Korea) and Eunjoon Kim (KAIST, Daejeon, South Korea), respectively.…”

“…CaMKIIα-SBFP2 was constructed by subcloning CaMKIIα from CaMKIIα-Venus (Addgene) by PCR in SBFP2-N1 vector. RNA interference-mediated nArgBP2 knockdown was carried out by expressing small hairpin RNA shRNA) duplexes in the pSiren-U6-mRFP vector (Clontech, Palo Alto, CA) and shRNA-resistant form of EGFP-nArgBP2 (henceforth, refer to as just EGFP-nArgBP2) was used as previously described ( Cho et al, 2023 ). Anti-HA frankenbody (15F11-HA scFv-mCherry, Plasmid #129591; Addgene) were used to label HA-SHANK3 for FRAP experiment.…”

“…Recent studies also demonstrate LLPS in additional post-synaptic proteins like GKAP, SHANK, Homer, and GluN2B ( Zeng et al, 2018 ). We recently also found that nArgBP2 undergoes LLPS both in vitro and in mature neurons, thereby regulating its function in the spatiotemporal control of structural synaptic plasticity ( Cho et al, 2023 ).…”