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

Takashi Kanadome,Natsumi Hoshino,Takeharu Nagai,Tomoki Matsuda,Takeshi Yagi

doi:10.1038/s41598-021-01481-2

Takashi Kanadome, Natsumi Hoshino + Show 3 more

Open Access

https://doi.org/10.1038/s41598-021-01481-2

Copy DOI

Abstract

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

Highlights

Clustered protocadherins (Pcdhs), which are cell adhesion molecules, play a fundamental role in self-recognition and non-self-discrimination by conferring diversity on the cell surface
Förster resonance energy transfer (FRET)-based Pcdh indicators, we used the protocadherin-γB2 isoform because structural information was available regarding a trans-dimer formed by longer ectodomain fragments (EC1-EC5) of γB2 (PDB: 5T9T)[27], which was helpful in determining the insertion sites of fluorescent proteins (FPs) for the FRET donor and acceptor
Since the low localization of γB2 at the plasma membrane hampers the development of FRET-based indicators for monitoring γB2 trans interactions across cells, we deleted the intracellular domain (ICD) to efficiently localize FP-inserted γB2s at the plasma membrane[33]

Summary

Introduction

Clustered protocadherins (Pcdhs), which are cell adhesion molecules, play a fundamental role in self-recognition and non-self-discrimination by conferring diversity on the cell surface. We developed the indicators by individually inserting FRET donor and acceptor fluorescent proteins (FPs) into the ectodomain of Pcdh molecules. We expect that FRET-based indicators for visualizing Pcdh trans interactions will provide a new approach for investigating the roles of Pcdh in self-recognition and non-self-discrimination processes. FRET donor and acceptor FPs are separately inserted into the ectodomain of N-cadherin molecules, which are close to the trans-interacting interface[32]. Using this FRET-based indicator for N-cadherin trans interaction, Kim et al investigated the C a2+-responsive dynamics of N-cadherin trans interaction across c ells[32]

Methods

Results

Conclusion