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

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.

Visualization oftranshomophilic interaction of clustered protocadherin in neurons

“…These results suggest that the combined γB2ΔICD-FRET and the combined γB2FL-FRET probe maintain their cis interaction properties with α4. As reported for the separated γB2-FRET probes (22), the FRET signals at the cell contact sites of the combined γB2ΔICD-FRET probe-expressing cells decreased upon EGTA addition (Figures 1K and 1L). These results indicate that the homophilic interaction of the γB2-FRET probe is extracellular calcium-dependent.…”

“…The combined γB2ΔICD-FRET probe in HEK293T cells revealed a FRET efficiency of 21.8% ± 6.06% (Figure 1I, mean ± SD). This is slightly lower than the separated γB2ΔICD-FRET probes (27.4 ± 5.67%) (22), but is high enough to analyze the homophilic interaction of γB2. It is possible that the increased FRET/CFP ratio at the cell contact sites is due to the change in environmental factors between the extracellular and intracellular regions, that a cis interaction at the cell contact sites could show FRET signals, and that FRET occurs intramolecularly at the cell contact sites.…”

“…Previously, we produced a set of FRET probes comprising γB2-EC1-CFP (CFP fused to extracellular domain 1 of PcdhγB2) and γB2-EC5-YFP (YFP fused to extracellular domain 5 of PcdhγB2): separated γB2-FRET probes (Figure 1A) (22). We prepared γB2-FRET probes with the intracellular domains (ICD) deleted because ICD deletion has been reported to result in efficient localization to the plasma membrane (23).…”

“…In neurons, homophilic interactions are implicated through self-crossing phenotypes in Pcdhγ KO mice (17)(18)(19) and overexpressing matched-/mismatched-isoforms (20,21); however, where and when the Pcdh homophilic interactions occur in neurons remains unknown. We previously reported a FRET probe for visualizing Pcdh homophilic interactions in cultured cell lines (22). Previous studies have shown that Pcdhγ proteins are localized at the contact sites of neurites and synapses (23)(24)(25)(26).…”

Development of intensiometric indicators for visualizing N-cadherin interaction across cells

“…Cell aggregation assay using K562 cells was performed as described previously 51 . Transfected K562 cells were rotated at 30 rpm overnight at 37 °C in humidified air containing 5% CO 2 .…”