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.2021 Nov 15;11(1):22237.
doi: 10.1038/s41598-021-01481-2.

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

Affiliations

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

Takashi Kanadome et al. Sci Rep..

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.

© 2021. The Author(s).

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Molecular design and cellular localization of FP-inserted γB2. (a) Schematics of full-length protocadherin-γB2 (γB2). γB2 is represented as serially repeated extracellular domains (EC domains), a transmembrane region (TM), and a cytoplasmic region (Cyto). The insertion positions (amino acid position 29th and 472nd residues in the matured form) of fluorescent protein (FP) are indicated by yellow arrowheads. (b) The localization of Venus-inserted γB2 constructs in HEK293T cells. HEK293T cells expressing the indicated constructs were observed using a confocal microscope. Fluorescence images (upper) and merged fluorescence and differential interference contrast (DIC) images (lower) are shown. Scale bar, 20 μm. (c) Schematics of FP-inserted γB2ΔICDs. In γB2ΔICD-EC1-mTQ2 and γB2ΔICD-EC5-Venus, mTurquoise2 (mTQ2) and Venus are inserted in γB2ΔICD at the positions as indicated in (a). ΔICD denotes deletion of an intracellular domain (ICD), which leads to efficient localization of γB2 at the plasma membrane. (d) The localization of FP-inserted γB2ΔICDs in HEK293T cells. HEK293T cells expressing the indicated constructs were observed using a confocal microscope. Scale bar, 20 μm.
Figure 2
Figure 2
The γB2trans interaction monitored by FRET-based γB2 indicators. (a) Schematics of the γB2trans interaction, as visualized using the FRET-based γB2 indicators. The orange ovals represent the EC domains of γB2. mTQ2 and Venus are represented as cyan and yellow cylinders, respectively. (b) Schematics of linker-optimized FRET-based γB2 indicators. The C-terminal 6 amino acids of mTQ2 are deleted, and a GGGGS (G4S) linker is inserted at the N-terminus of mTQ2 in γB2ΔICD-EC1-G4S-mTQ2ΔC6. The 3 N-terminal amino acids and the 9 C-terminal amino acids of Venus are deleted, and a 3xPro (P3) linker is inserted at the C-terminus of Venus in γB2ΔICD-EC5-VenusΔN3C9-P3. (c) Representative acceptor photobleaching experiments of the pre- and post-optimized FRET-based γB2ΔICD indicators in HEK293T cells. HEK293T cells individually expressing the indicated constructs were co-cultured (left panels). The panels on the right represent magnified fluorescence images of white squared regions in left panels before and after acceptor photobleaching at green circles. The color bars indicate the range of the fluorescence intensities of mTQ2 and Venus (arbitrary units). Scale bars, 20 μm (left panel) and 2 μm (right panel). (d) Alterations in fluorescence intensities of mTQ2 and Venus by acceptor photobleaching in (c) were followed and plotted over time. (e) Mean values of FRET efficiency for the pre- and post-optimized FRET-based γB2ΔICD indicators (Pre,n = 28; Post,n = 29). Data are shown as the means ± SD. Significant difference was analyzed by Welch’st test.p value is described in the graph. (f) Binding specificity of the FRET-based γB2ΔICD indicators by cell aggregation assay. K562 cells individually expressing the indicated constructs were co-cultured. Scale bar, 200 μm. (g) The interaction between γB2ΔICD-EC1-mTQ2ΔC6 and γB2ΔICD-EC5-VenusΔN3C9-P3, as revealed by cell aggregation assay. K562 cells individually expressing the indicated constructs were co-cultured. Scale bar, 200 μm. (h) Direct visualization of the γB2trans interaction using the FRET-based γB2ΔICD indicators. K562 cells individually expressing the indicated constructs were co-cultured. The merged fluorescence and DIC image is shown in the left panel. The ratio image of Venus/mTQ2 is shown as intensity-modulated display (IMD) in the right panel. Scale bar, 10 μm.
Figure 3
Figure 3
Specificity of FRET-based γB2ΔICD indicators. (a) Schematics of FRET-based chimeric γB2γA3ΔICD indicators. The EC2-EC3 domains of the FRET-based γB2ΔICD indicators were swapped by those of γA3. The orange and light blue boxes are the EC domains from γB2 and γA3, respectively. (b) The localization of the FRET-based chimeric γB2γA3ΔICD indicators in HEK293T cells. Scale bar, 20 μm. (c) Binding specificity of the FRET-based chimeric γB2γA3ΔICD indicators, as revealed by cell aggregation assay. K562 cells individually expressing the indicated constructs were co-cultured. Scale bar, 200 μm. (d) Evaluation of the FRET-based chimeric γB2γA3ΔICD indicators and the FRET-based γB2ΔICD indicators by acceptor photobleaching in HEK293T cells. Acceptor photobleaching at the cell adhesion sites was performed using co-cultured HEK293T cells in the indicated combination, and FRET efficiency was calculated [n = 20 (γB2–γB2),n = 20 (γB2γA3–γB2γA3),n = 18 (γB2–γB2γA3),n = 19 (γB2γA3–γB2). The combination is described as mTQ2-Venus pair]. Data are shown as the means ± SD. Significant differences were analyzed by Welch’s ANOVA test, followed by Dunnett’s T3 multiple comparison test.p values are indicated in the graph. (e) Cell aggregation between the FRET-based γB2ΔICD- and chimeric γB2γA3ΔICD indicator-expressing cells, mediated by co-expressing N-cadherin. K562 cells individually expressing the indicated FRET constructs and N-cadherin were co-cultured. Scale bar, 200 μm. (f) FRET between the FRET-based γB2ΔICD- and chimeric γB2γA3ΔICD indicators. K562 cells expressing N-cadherin and the FRET-based γB2ΔICD- or the chimeric γB2γA3ΔICD indicators were co-cultured in the indicated combination. The merged fluorescence and DIC images are shown in the upper panels. The ratio images of Venus/mTQ2 are shown as IMD in the lower panels. mTQ2- and Venus-positive cell adhesion sites are indicated by white arrowheads. Scale bar, 10 μm.
Figure 4
Figure 4
Ca2+-dependency of γB2trans interaction. (a) The effect of EGTA treatment on the FRET at the cell adhesion sites. K562 cells individually expressing the FRET-based γB2ΔICD indicators were co-cultured. The merged fluorescence and DIC images (upper panels) and the ratio images of Venus/mTQ2 (lower panels) were acquired before and after 5 mM EGTA treatment. Scale bar, 10 μm. (b) Change in Venus/mTQ2 ratio (ΔR/R0) at a cell adhesion site with EGTA treatment over time. EGTA (5 mM) was added to the medium at 30 s. R0 was calculated as the average Venus/mTQ2 ratio value of 30 frames before EGTA treatment. (c) Emission spectra before and after EGTA treatment. The emission spectra excited by 405 nm light at the cell adhesion site before and after 5 mM EGTA treatment were acquired. (d) Quantitative analysis of ratio changes by EGTA treatment. The FRET ratio values at the cell adhesion sites before and after 5 mM EGTA treatment were measured (n = 38). Significant difference was analyzed by Wilcoxon matched-pairs signed rank test.p value is indicated in the graph.
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