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Development of FRET‑based indicators for visualizing homophilic trans interaction of a clustered protocadherin

星野, 七海 大阪大学 DOI:10.18910/88168

2022.03.24

概要

Clustered protocadherins (Pcdhs), which are cell adhesion molecules, play a fundamental role in self- recognition and non-self-discrimination by conferring diversity to the cell surface. Although systematic cell-based aggregation assays provide information regarding the binding properties of Pcdhs, the direct visualization of homophilic Pcdh trans interactions across cells remains challenging. Here, I present Förster resonance energy transfer (FRET)-based indicators for the direct visualization of homophilic Pcdh trans interactions. I developed the indicators by individually inserting FRET donor and acceptor fluorescent proteins (FPs) into the ectodomain of Pcdh molecules. This process enabled the successful visualization of specific homophilic Pcdh trans interactions and revealed that the homophilic Pcdh trans interaction is highly sensitive to changes in extracellular Ca2+ levels.

The developed FRET indicators did not exhibit any FRET signals at the contact sites when the acceptor and donor indicators were coexpressed in the cells. To enable the FRET-based visualization of the homophilic Pcdh trans interaction in self-neurites, I fused two FPs into a single Pcdh molecule, which means that the two indicators are united into a single indicator in which the donor and acceptor proteins are combinatorially fused. This “combined” FRET indicator successfully showed FRET signals at the cell–cell contact sites, but no FRET signals were revealed in the intracellular spaces.

Although two FPs were fused in the extracellular (EC) domains of Pcdh, cell aggregation assays and immunoprecipitation revealed that the FP-fused Pcdh exhibited normal binding affinity to other proteins that have been reported to interact with the EC domains of Pcdhs.

Furthermore, I constructed a transgenic mouse that expressed the combined FRET indicator in a Cre-dependent manner. In this mouse, I revealed that a single isoform of Pcdhγ can rescue the self-crossing phenotype in the dendrites of Purkinje cells with Pcdhγ deletion.

I expect that FRET-based indicators for visualizing homophilic Pcdh trans interactions will provide a new approach for investigating the roles of Pcdh in self-recognition and non-self- discrimination processes, including the regulation of synaptic specificity.

In this thesis, I explain why I developed an indicator of the homophilic trans interaction of Pcdh. Then, the results of the separated FRET indicators and those of a combined FRET indicator are presented. The results of the separated FRET indicators are from a previous study, of which I was a co-first author (Kanadome et al., 2021). Finally, I discuss the usage of these indicators, including the advantages and disadvantages of the two concepts.

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