KOKORO-Biology Group, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan.
Department of Biomolecular Science and Engineering, SANKEN, Osaka University, Ibaraki, Osaka 567-0047, Japan.
Proc Natl Acad Sci U S A. 2023 Sep 19;120(38):e2301003120. doi: 10.1073/pnas.2301003120. Epub 2023 Sep 11.
Clustered protocadherin (Pcdh) functions as a cell recognition molecule through the homophilic interaction in the central nervous system. However, its interactions have not yet been visualized in neurons. We previously reported PcdhγB2-Förster resonance energy transfer (FRET) probes to be applicable only to cell lines. Herein, we designed γB2-FRET probes by fusing FRET donor and acceptor fluorescent proteins to a single γB2 molecule and succeeded in visualizing γB2 homophilic interaction in cultured hippocampal neurons. The γB2-FRET probe localized in the soma and neurites, and FRET signals, which were observed at contact sites between neurites, eliminated by ethylene glycol tetraacetic acid (EGTA) addition. Live imaging revealed that the FRET-negative γB2 signals rapidly moved along neurites and soma, whereas the FRET-positive signals remained in place. We observed that the γB2 proteins at synapses rarely interact homophilically. The γB2-FRET probe might allow us to elucidate the function of the homophilic interaction and the cell recognition mechanism.
簇状原钙黏蛋白 (protocadherin, Pcdh) 通过在中枢神经系统中的同种型相互作用发挥细胞识别分子的功能。然而，其相互作用尚未在神经元中可视化。我们之前报道过 PcdhγB2-荧光共振能量转移（Förster resonance energy transfer, FRET）探针仅适用于细胞系。在此，我们通过将 FRET 供体和受体荧光蛋白融合到单个γB2 分子上设计了γB2-FRET 探针，并成功地在培养的海马神经元中可视化了γB2 同种型相互作用。γB2-FRET 探针定位于神经元的胞体和突起中，并且在突起之间的接触部位观察到的 FRET 信号可以被乙二胺四乙酸（ethylene glycol tetraacetic acid, EGTA）消除。活细胞成像显示，FRET 阴性的γB2 信号沿着突起和胞体快速移动，而 FRET 阳性信号则保持原位。我们观察到突触处的γB2 蛋白很少发生同种型相互作用。γB2-FRET 探针可能使我们能够阐明同种型相互作用和细胞识别机制的功能。
