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

nArgBP2, 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.

Multivalent electrostatic pi-cation interaction between synaptophysin and synapsin is responsible for the coacervation.

We recently showed that synaptophysin (Syph) and synapsin (Syn) can induce liquid-liquid phase separation (LLPS) to cluster small synaptic-like microvesicles in living cells which are highly reminiscent of SV cluster. However, as there is no physical interaction between them, the underlying mechanism for their coacervation remains unknown. Here, we showed that the coacervation between Syph and Syn is primarily governed by multivalent pi-cation electrostatic interactions among tyrosine residues of Syph C-terminal (Ct) and positively charged Syn. We found that Syph Ct is intrinsically disordered and it alone can form liquid droplets by interactions among themselves at high concentration in a crowding environment in vitro or when assisted by additional interactions by tagging with light-sensitive CRY2PHR or subunits of a multimeric protein in living cells. Syph Ct contains 10 repeated sequences, 9 of them start with tyrosine, and mutating 9 tyrosine to serine (9YS) completely abolished the phase separating property of Syph Ct, indicating tyrosine-mediated pi-interactions are critical. We further found that 9YS mutation failed to coacervate with Syn, and since 9YS retains Syph's negative charge, the results indicate that pi-cation interactions rather than simple charge interactions are responsible for their coacervation. In addition to revealing the underlying mechanism of Syph and Syn coacervation, our results also raise the possibility that physiological regulation of pi-cation interactions between Syph and Syn during synaptic activity may contribute to the dynamics of synaptic vesicle clustering.

Cooperative function of synaptophysin and synapsin in the generation of synaptic vesicle-like clusters in non-neuronal cells.

Clusters of tightly packed synaptic vesicles (SVs) are a defining feature of nerve terminals. While SVs are mobile within the clusters, the clusters have no boundaries consistent with a liquid phase. We previously found that purified synapsin, a peripheral SV protein, can assemble into liquid condensates and trap liposomes into them. How this finding relates to the physiological formation of SV clusters in living cells remains unclear. Here, we report that synapsin alone, when expressed in fibroblasts, has a diffuse cytosolic distribution. However, when expressed together with synaptophysin, an integral SV membrane protein previously shown to be localized on small synaptic-like microvesicles when expressed in non-neuronal cells, is sufficient to organize such vesicles in clusters highly reminiscent of SV clusters and with liquid-like properties. This minimal reconstitution system can be a powerful model to gain mechanistic insight into the assembly of structures which are of fundamental importance in synaptic transmission.

Severe Fever with Thrombocytopenia Syndrome Virus Infection or Mixed Infection with Scrub Typhus in South Korea in 2000-2003.

Severe fever with thrombocytopenia syndrome is a tick-borne viral disease, with a high mortality rate that was first reported in China in 2009. Scrub typhus is an acute febrile illness caused by Orientia tsutsugamushi, a bacterium transmitted to humans through chigger mite bites. Severe fever with thrombocytopenia syndrome and scrub typhus are endemic to South Korea. To investigate evidence of severe fever with thrombocytopenia syndrome virus (SFTSV) infection or mixed infection with scrub typhus in South Korea, we examined 2,329 sera samples collected from patients presenting from November 1, 2000, to November 1, 2003, for the diagnosis of rickettisal diseases at Seoul National University, Seoul, South Korea. We found retrospective evidence of SFTSV infection or mixed infection with scrub typhus in South Korea in 2000-2003. Severe fever with thrombocytopenia syndrome virus infections in South Korea occurred before previously reported cases and were more concurrent with those in China. It is important to consider SFTSV infection in patients with scrub typhus.

p21(WAF¹/C¹P¹) deficiency induces mitochondrial dysfunction in HCT116 colon cancer cells.

p21(WAF1/CIP1) is a critical regulator of cell cycle progression. However, the role of p21 in mitochondrial function remains poorly understood. In this study, we examined the effect of p21 deficiency on mitochondrial function in HCT116 human colon cancer cells. We found that there was a significant increase in the mitochondrial mass of p21(-/-) HCT116 cells, as measured by 10-N-nonyl-acridine orange staining, as well as an increase in the mitochondrial DNA content. In contrast, p53(-/-) cells had a mitochondrial mass comparable to that of wild-type HCT116 cells. In addition, the expression levels of the mitochondrial biogenesis regulators PGC-1α and TFAM and AMPK activity were also elevated in p21(-/-) cells, indicating that p21 deficiency induces the rate of mitochondrial biogenesis through the AMPK-PGC-1α axis. However, the increase in mitochondrial biogenesis in p21(-/-) cells did not accompany an increase in the cellular steady-state level of ATP. Furthermore, p21(-/-) cells exhibited significant proliferation impairment in galactose medium, suggesting that p21 deficiency induces a defect in the mitochondrial respiratory chain in HCT116 cells. Taken together, our results suggest that the loss of p21 results in an aberrant increase in the mitochondrial mass and in mitochondrial dysfunction in HCT116 cells, indicating that p21 is required to maintain proper mitochondrial mass and respiratory function.