Genetically Encoded Fluorescent Indicator GRAPHIC Delineates Intercellular Connections

Abstract

Intercellular contacts are essential for precise organ morphogenesis, function, and maintenance; however, spatiotemporal information of cell-cell contacts or adhesions remains elusive in many systems. We developed a genetically encoded fluorescent indicator for intercellular contacts with optimized intercellular GFP reconstitution using glycosylphosphatidylinositol (GPI) anchor, GRAPHIC (GPI anchored reconstitution-activated proteins highlight intercellular connections), which can be used for an expanded number of cell types. We observed a robust GFP signal specifically at the interface between cultured cells, without disrupting natural cell contact. Application of GRAPHIC to the fish retina specifically delineated cone-bipolar connection sites. Moreover, we showed that GRAPHIC can be used in the mouse central nervous system to delineate synaptic sites in the thalamocortical circuit. Finally, we generated GRAPHIC color variants, enabling detection of multiple convergent contacts simultaneously in cell culture system. We demonstrated that GRAPHIC has high sensitivity and versatility, which will facilitate the analysis of the complex multicellular connections without previous limitations.

Keywords: Biological Sciences; Cell Biology; Molecular Biology.

Graphical abstract

Figure 1

Design of GRAPHIC and Its Signal Pattern (A) Diagram of GRAPHIC molecular structures. GRAPHIC molecules consist of signal peptide (SP), split sfGFP fragment, leucine zipper domains (LZA or LZB), and GPI anchor domain. To identify and estimate probe expression, H2B-mCherry and H2B-Azurite were co-expressed with NT-probe and CT-probe, respectively. (B) GFP molecule consists of 11 β-sheets and fluorophore domain (FL). sfGFP split site for GRAPHIC is between seventh and eighth β-sheet (7/8). Other intercellular probe systems utilize 10/11 split site. (C) Diagram of GRAPHIC labeling intercellular contact. GFP molecules are reconstituted by intercellular interaction of a set of probe molecules at cell-cell contact site.

Figure 2

Comparison of GRAPHIC with Other Probes (A) GRAPHIC showed higher signal intensity in LLCPK1 epithelial cell culture. Modification of GFP split site (10/11 to 7/8) in mGRASP system increased its signal intensity. All three culture and image acquisition conditions are same. (B) Quantification and comparison of GRAPHIC (n = 57) and modified mGRASP (7/8) (n = 66) signal intensity. GFP signals were normalized with co-expressed nuclei label intensities of contacted cells. GFP-NT fragment (post-mGRASP and n-GRAPHIC)-expressing cells are red nuclei (H2B-mCherry, attached with 2A peptide) and GFP-CT fragment (pre-mGRASP and c-GRAPHIC)-expressing cells are blue nuclei (H2B-Azurite, attached with 2A peptide). ∗∗∗p = 1.09 × 10⁻²⁴; Student's unpaired t test. Scale bars, 20 μm.

Figure 3

Characteristics of GRAPHIC Signal (A and B) Time-lapse images of GRAPHIC signal in constructing (A) or disrupting (B) intercellular contact between n-GRAPHIC-expressing LLCPK1 cells (red nuclei) and c-GRAPHIC-expressing LLCPK1 cells (blue nuclei). Upper panels are merged images of bright-field (differential interference contrast), RFP and BFP fluorescence. Bottom panels are GFP fluorescent images. In (A), two cell lines first contacted at time 0. In (B), EDTA was administrated at time 0 (final concentration; 5mM). (C) Quantification of relative membrane retraction velocity during 10–12 min after EDTA ion chelation. H2B-mCherry-expressing (without GRAPHIC) LLCKP1 cells (n = 51) were used as control. Membrane retraction velocity of GRAPHIC was calculated between n- and c-GRAPHIC-expressing cells (n = 33). Student's unpaired t test. (D-F) GRAPHIC signal still remains in completely dissociated cells. Dissociated (with 5 mM EDTA, without trypsin) LLCPK1 cells from single culture of RFP+ (n-GRAPHIC expressing) or BFP+ (c-GRAPHIC expressing) cell line and co-culture of both cell lines were subjected to flow cytometry (single cultured RFP+; n = 4751, single cultured BFP+; n = 4851, co-cultured RFP+; n = 6062, co-cultured BFP+; n = 5131). Both microscope observation (D and E) and histogram of GFP intensity (F) of sorted cells showed co-culture dependent GRAPHIC signal in dissociated single cell. Scale bars, 20 μm.

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.

Figure 5

GRAPHIC Visualizes Synaptic Connection in Neuronal Networks (A and B) GRAPHIC labels synaptic sites in zebrafish retina. (A) In the zebrafish retina, typical structures, ribbon synapses, are formed between cone photoreceptor axon terminals, namely, pedicles and dendrites of bipolar cells. n-GRAPHIC and membrane-targeted tdTomato were co-expressed in a subpopulation of bipolar cells using the vsx1 promoter (vsx1: memtdTomato-2A-n-GRAPHIC), and c-GRAPHIC and TagBFP2 were co-expressed in cone cells using the guanine nucleotide-binding protein G(t) subunit alpha-2 (gnat2) gene promoter (gnat2: TagBFP2-2A-c-GRAPHIC). Both expression vectors were co-injected into one-cell-stage zebrafish embryos and then fixed at 5 days postfertilization. (B) Reconstituted GFP signals were only detected at the dendritic tips of tdTomato-positive bipolar cell closely associated with TagBFP2-expressing cone pedicle. Scale bar, 5 μm. (C–E) GRAPHIC signals in S1 layer IV neurons are merged with thalamocortical axons of VB neurons in mouse brain. (C) In utero electroporation (IUE) was performed at E13.5–14.5 to express n-GRAPHIC-2A-H2B-mCherry in cortical layer IV. After the electroporated mice had grown up to P14-21, c-GRAPHIC-2A-mCherry encoding AAV was stereotaxically injected into VB. After 30–40 days, the injected brains were sectioned and observed. (D) Certain number of cortical layer IV neurons (bracket) expressed n-GRAPHIC (indicated by co-expressed red nuclei label, dot line) and many mCherry labeled fibers of c-GRAPHIC expressing VB neurons reached to cortical layer IV (white arrows). (E) Higher magnification of confocal images of layer IV showed many GFP puncta largely overlapped with mCherry-positive bouton-like structures. GFP puncta that co-localize with mCherry-positive axons are indicated by arrowheads. Scale bars, 200 μm in (D) and 5 μm in (E). (F) IUE was performed at E13.5 to express n-GRAPHIC-2A-PSD95-mCherry in cortical layer IV. After the electroporated mice had grown to adult (about 2-month-old), c-GRAPHIC-2A-H2B-mCherry encoding AAV was stereotaxically injected into thalamus VB. After about 4 weeks, the injected brains were sectioned and observed. (G) Distribution of GRAPHIC signals in cortical layer IV neurons. GFP (GRAPHIC) signals indicate contacted sites between n-GRAPHIC-expressing layer IV neurons and c-GRAPHIC-expressing VB neurons. PSD95-mCherry signals indicate postsynaptic sites of electroporated layer IV neurons. GFP puncta that co-localize with PSD95-mCherry puncta are indicated by arrowheads. Scale bar, 5 μm.