Visualization of trans homophilic interaction of clustered protocadherin in neurons

Clustered protocadherin (Pcdh) functions as a cell recognition molecule through the homophilic interaction in CNS. However, its interactions have yet not been visualized in neurons. We previously reported Pcdh{gamma}B2-FRET probes to be applicable only for cell lines. Herein, we newly designed Pcdh{gamma}B2-FRET probes by fusing FRET donor and acceptor fluorescent proteins to a single Pcdh{gamma}B2 molecule and succeeded in visualizing Pcdh{gamma}B2 homophilic interaction in cultured hippocampal neurons. The {gamma}B2-FRET probe localized in the soma and neurites, and FRET signals were observed at contact sites between neurites and eliminated by EGTA addition. Live imaging revealed that the FRET-negative {gamma}B2 signals were rapidly moving along neurites and soma, whereas the FRET-positive signals remained in place. We observed that the {gamma}B2 proteins at synapses rarely interact homophilically. The {gamma}B2-FRET probe would allow us to elucidate the function of the homophilic interaction and the cell recognition mechanism. Significance StatementWe visualize the Pcdh homophilic interaction using a novel FRET-based probe, and reveal that the homophilically interacting Pcdh proteins are found at contact sites between the neurites and roots of neurites from the soma, and are stable at a location. Additionally, in neurons, Pcdh proteins are located at synapses but rarely interact homophilically.