Figure 4.

Development of Color Variants of GRAPHIC

(A–F) Fluorescent character of BFP is mainly dependent on GFP-NT fragment region (GFP 1-7). Substitution of eight amino acid residues in GFP 1-7, I39N, T65S, Y66H, S72A, K105T, T128V, V150I, D155V altered the fluorescent character of reconstituted signal, GFP to BFP. GFP-type combination (GN + GC) signal at cell-cell contact sites of LLCPK1 could be detected with microscope filter set for GFP detection (excitation 465-485 nm, emission 502-534 nm) (C), but not for BFP (excitation 355-405 nm, emission 420-480 nm) (B), whereas BFP-type combination (BN + GC) signal could be detected with microscope filter set for BFP (E), but not for GFP (F).

(G and H) Fluorescent character of YFP is mainly dependent on T203I amino acid substitution of GFP, which is within GFP-CT fragment region (GFP 8-11). (H) Comparing fluorescent spectrums at cell-cell contact sites of GFP- (GN + GC) and YFP-type combination (GN + YC), T203I substitution in GFP 8-11 domain of c-GRAPHIC shifted reconstituted signal character to YFP-like longer wavelength. Error bars, ± SD.

(I–N) Co-culture of three LLCPK1 cell lines (GN cells express n-GRAPHIC and H2B-mCherry, GC cells express only c-GRAPHIC, and YC cells express YFP type c-GRAPHIC and H2B-Azurite) showed that the GRAPHIC system simultaneously detected multiple connectivity in one cell. (I) GN* cell contacts with both GC cell and YC cell. (J) Fluorescent spectra at points 1 and 2 showed GFP-like and YFP-like characters, respectively. (K) Ratiometric image of reconstituted signal intensities at 510 nm (gated 505–515 nm) (L) and 525 nm (gated 520–530 nm) (M). GN* cell contacts with both GC cell and YC cell, and each contact region can be separated by its fluorescent character (K and N). Scale bars, 20 μm.