Fluorescent indicators for visualizing dynamic contact between cells and between processes originating from a single cell

Takashi Kanadome; Natsumi Hoshino; Susumu Jitsuki; Hidehiko Hashimoto; Takeshi Yagi; Takeharu Nagai

doi:10.1016/j.crmeth.2025.101292

. 2026 Jan 23;6(2):101292. doi: 10.1016/j.crmeth.2025.101292

Fluorescent indicators for visualizing dynamic contact between cells and between processes originating from a single cell

Takashi Kanadome ^1,^2,^3,^4,^5,^6,^12,^13,^∗, Natsumi Hoshino ^7,^8,¹², Susumu Jitsuki ⁹, Hidehiko Hashimoto ^1,¹⁰, Takeshi Yagi ⁷, Takeharu Nagai ^6,¹¹

PMCID: PMC12946757 PMID: 41579865

Summary

Cells continuously communicate through dynamic cell-cell contacts. Tools for visualizing these dynamic interactions in living cells are essential to the study of fundamental biological processes in multicellular organisms. Here, we present two fluorescent indicators, Gachapin and Gachapin-C, for visualizing dynamic cell-cell contact. Gachapin visualizes not only static but also dynamic contacts. Multiplexed imaging combining green Gachapin with spectrally distinct indicators allows simultaneous monitoring of contact dynamics, cytoskeletal assembly, and intracellular signaling during cell movement. Furthermore, the formation and disruption of contacts between neuronal processes can be visualized. Gachapin-C enables contact visualization with a single indicator component, whereas previous indicators required two components introduced into different cells. This feature allows Gachapin-C to monitor contacts between processes originating from a single cell. We expect Gachapin and Gachapin-C will serve as useful tools for providing deeper insights into cell-cell contact-mediated processes.

Keywords: fluorescent indicator, ddGFP, bioimaging, cell-cell contact, neuronal self-recognition

Graphical abstract

Highlights

•
Gachapin is a reversible fluorescent indicator for dynamic cell-cell contacts
•
Gachapin enables multiplexed imaging of contact, actin, and signaling dynamics
•
Gachapin-C is a single-component indicator for cell-cell and self-contacts
•
Gachapin-C can monitor contacts between processes originating from a single cell

Motivation

Fluorescent indicators based on dimerization-dependent fluorescent proteins (ddFPs) have been developed for visualizing specific interactions of cell adhesion molecules between cells. While these allow real-time imaging in contrast to previous indicators based on irreversible split-GFP, fluorescent indicators that allow visualization of simple cell-cell contacts are currently limited. Here, we present two ddFP-based fluorescent indicators, Gachapin and Gachapin-C, for visualizing such cell-cell contacts rather than specific interactions of cell adhesion molecules.

Kanadome et al. develop Gachapin and Gachapin-C, two reversible indicators for cell-cell contact dynamics. Gachapin enables multiplexed imaging of intercellular contacts alongside cytoskeletal and signaling activities. Gachapin-C provides more specific visualization of self-contacts within a single cell. These tools reveal contact assembly and disassembly in real time.

Introduction

In multicellular organisms, cells communicate with each other by direct contact. Dynamic cell-cell contact is involved in a wide range of biological processes, such as contact inhibition of locomotion,¹ cell differentiation during ontogeny,² activation and maturation of immune cells,³ axonal and dendritic tiling in neurons,⁴ and neuronal synapse formation.⁵ To study and understand cell-cell contact and biological phenomena mediated by cell-cell contact, fluorescent indicators for visualizing cell-cell contact have been developed, which are classically based on a split-GFP technique.⁶^,⁷ The split-GFP technique was originally developed to visualize protein-protein interactions in cells. Non-fluorescent fragments obtained by dividing GFP into two parts are fused to target proteins, and the fluorescence is emitted by the reconstitution of GFP fragments upon interaction between the target proteins. Split-GFP-based indicators in which split GFP fragments are localized at the cell surface have allowed the visualization of cell-cell contact, especially neuronal connections.⁸^,⁹^,¹⁰^,¹¹^,¹²^,¹³ Although they are potent tools to visualize static cell-cell contact, their time delay of fluorescence emission derived from chromophore maturation and their irreversible nature hamper the real-time visualization of dynamic cell-cell contact.

To address this problem, we have developed another type of indicator based on a dimerization-dependent fluorescent protein (ddFP) technique. ddFP consists of two components, ddFP-A and ddFP-B, which have and lack a chromophore, respectively. ddFP-A is quenched in the monomeric state but emits fluorescence upon heterodimerization with ddFP-B, which is a reversible process.¹⁴ A red-colored ddRFP was originally developed from dTomato, and then a green-colored ddGFP was created.¹⁵ Owing to the reversible nature of ddGFP, we have previously developed fluorescent indicators, namely INCIDER and IPAD, to visualize reversible interactions of specific cell adhesion molecules, N-cadherin and clustered protocadherin-α4 (Pcdhα4), between cells.¹⁶^,¹⁷ cIPAD, an IPAD variant, successfully visualized Pcdhα4 interactions between processes originating from the same cell.¹⁷^,¹⁸ In addition, SynapShot based on the ddFP technique has recently been developed by another group to visualize reversible interactions between neurexin and neuroligin at the neuronal synapses.¹⁹ Thus, our group and another group have clearly demonstrated the superiority of the ddFP technique over the split-GFP technique in terms of reversibility. Although these indicators allow us to visualize reversible interactions of specific cell adhesion molecules across membranes, no fluorescent indicators to visualize simple cell-cell contact, rather than interactions of specific cell adhesion molecules, have yet been reported.

In this study, we report the development of a fluorescent indicator using ddGFP to visualize simple cell-cell contact, rather than interactions of specific cell adhesion molecules. The indicator, namely Gachapin, allows the visualization of not only static cell-cell contact but also dynamic cell-cell contact. Multiplexed imaging of Gachapin enables simultaneous monitoring of cell-cell contact dynamics, F-actin dynamics, and Rho-family small GTPase activity during cell movement. The formation and disruption of contact between processes of neurons can also be visualized. We also report the development of another fluorescent indicator based on ddGFP, namely Gachapin-C. Although previous indicators, including Gachapin, require two indicator components to be expressed separately in discrete cells in order to visualize cell-cell contact, Gachapin-C allows visualization of cell-cell contact with only one indicator component. This property of Gachapin-C also allows monitoring of contact between processes originating from the same cell.

Results

Design and characterization of the fluorescent indicator for visualizing cell-cell contact, termed Gachapin

We previously developed fluorescent indicators for visualizing trans-interactions of specific cell adhesion molecules with a reversible property using ddGFP.¹⁶^,¹⁷^,¹⁸ To develop a fluorescent indicator for visualizing simple cell-cell contact, rather than specific protein interactions, with a reversible property, we decided to design the indicator to expose ddGFP-A and ddFP-B on different cell surfaces, respectively. ddFP-B has two variants with different affinities to ddGFP-A, namely ddFP-B1 (K_D = 3 μM) and ddFP-B3 (K_D = 40 μM).²⁰ Since eGRASP¹¹ and GRAPHIC,¹² split-GFP-based indicators for visualizing cell-cell interactions, have succeeded in enhancing their fluorescence intensities by increasing the affinity between indicator components through the introduction of trans-interacting motifs on each indicator component, we used ddFP-B1 as ddFP-B with high affinity for ddGFP-A. In addition, we also tried to prepare constructs containing two ddGFP-A and ddFP-B1 in tandem to increase their local concentration. GRAPHIC efficiently visualized cell-cell interactions by using a GPI anchor to localize to the plasma membrane. Furthermore, eLACCO1.1,²¹ a fluorescent indicator for visualizing extracellular L-lactate, was optimized for its performance by combining a GPI anchor to localize at the plasma membrane and a 218 linker that was reported to reduce aggregation and enhance proteolytic stability in single-chain Fv.²² On the basis of these notions, we prepared four indicator variants, termed GPI-anchored cell-cell contact indicator by heterodimerization of ddGFP-A and ddFP-B proteins (Gachapin). Gachapin consists of two components, ddGFP-A and ddFP-B constructs, named Gachapin-A and Gachapin-B, respectively (Figure 1A). To examine their performance, we co-cultured HEK293T cells individually expressing Gachapin components. All Gachapin variants emitted green fluorescence at the cell-cell contact sites (Figure 1B). Quantification of Gachapin signals at the cell-cell contact sites showed that Gachapin-4 visualized cell-cell contact with the highest brightness of the four Gachapin variants (Figure 1C). Hereafter, we used Gachapin-4 as Gachapin. It is possible that Gachapin expression induces cell adhesion. To examine this possibility, we performed a cell aggregation assay using K562 cells. K562 cells lack endogenous cell adhesion molecules and have been widely used to study the cell adhesion function of exogenous cell adhesion molecules.²³^,²⁴^,²⁵^,²⁶^,²⁷^,²⁸ The cell aggregation assay showed that Gachapin-A-expressing cells and Gachapin-B-expressing cells rarely formed co-aggregates, similar to mCherry-NLS-expressing cells and EBFP2-NLS-expressing cells, although N-cadherin (NCad)-expressing cells efficiently formed cell aggregates (Figure 1D). CoAggregation (CoAg) Index,²⁹^,³⁰ a metric to quantify the degree of co-aggregation, clearly showed that Gachapin did not induce cell aggregation, in contrast to NCad (Figure 1E). These results indicate that Gachapin is able to visualize cell-cell contact without inducing cell adhesion.

Development and characterization of Gachapin

(A) Schematics of a series of Gachapin variants. Gachapin consists of two components, Gachapin-A and Gachapin-B. SS, GPI, and 218 represent an acrosin-derived signal sequence, a Thy-1 N-terminal GPI-linked signal sequence, and a flexible peptide linker whose sequence is GSTSGSGKPGSGEGSTKG, respectively.

(B) Visualization of static cell-cell contact using Gachapin variants. HEK293T cells individually expressing Gachapin components were co-cultured in the indicated combinations and then observed using a confocal microscope. To label cells expressing Gachapin-A and -B, mCherry and EBFP2 fused to an SV40-derived nuclear localization signal (NLS), i.e., mCherry-NLS and EBFP2-NLS, were co-expressed bicistronically via a self-cleavable P2A peptide, respectively. Scale bars, 10 μm. Fluorescence profiles on the yellow line from X to X′ are shown on the right. Relative fluorescence intensities of EBFP2-NLS, Gachapin, and mCherry-NLS are represented by blue, green, and red lines, respectively.

(C) Comparison of brightness among Gachapin variants. Fluorescence intensity of Gachapin at the cell contact sites was quantified and compared among the variants. 77 (Gachapin-1), 118 (Gachapin-2), 165 (Gachapin-3), and 162 (Gachapin-4) cell-cell contact sites from six independent cultures were analyzed. The plot shows data from all individual cell-cell contact sites (gray dots). The mean value from each independent culture is overlaid as a red dot. The final value is presented as the mean ± SD of these independent culture means. Statistical significance was assessed by one-way ANOVA, followed by Tukey’s multiple comparison test using mean values of six independent cultures.

(D) The effect of Gachapin expression on cell adhesion. K562 cells individually expressing the indicated constructs were co-cultured and observed using a confocal microscope. Scale bars, 100 μm.

(E) CoAg Index was calculated based on (D). 30 images from three independent cultures were analyzed. The plot shows data from all individual images (gray dots). The mean value from each independent culture is overlaid as a red dot. The final value is presented as the mean ± SD of these independent culture means. Statistical significance was assessed by one-way ANOVA, followed by Tukey’s multiple comparison test using mean values of three independent cultures.

Visualization of dynamic cell-cell contact by Gachapin

To investigate the detectability of dynamic cell-cell contact by Gachapin, we observed co-cultured HEK293A cells individually expressing Gachapin components over time. We chose to use this cell line for this experiment because, in our experience, they are highly motile. Time-lapse imaging showed that the formation and disruption of cell-cell contact accompanied by cell movement were visualized by Gachapin (Figures 2A and S1; Video S1). We likewise examined a split-GFP-based indicator by imaging co-cultured HEK293A cells expressing mGRASP⁹ components over time. In contrast to Gachapin, the formation and disruption of cell-cell contact were not observed with mGRASP (Figure S2; Video S2). These observations indicate that Gachapin can visualize dynamic cell-cell contact that is not captured by split-GFP-based indicators. Cell movement is regulated by actin remodeling driven by Rho-family GTPases such as Rac1 and RhoA.³¹^,³² To visualize cell-cell contact, actin dynamics, and Rho GTPase activity simultaneously, we aimed to use Lifeact³³ fused to EBFP2 for actin, green Gachapin for contact, and ddRFP-based indicators R-Rac1 and R-RhoA. While ddFP-based Rac1 indicators³⁴ were well characterized, the ddFP-based RhoA indicators³⁵ required further validation. We initially tested the ddGFP-based RhoA indicators (G-RhoA) using constitutively active (Q63L) and dominant-negative (T19N) mutants (Figure S3A). Although T19N fluorescence was weaker than that of Q63L, both mutants emitted fluorescence, indicating poor signal contrast (Figure S3B). This finding is consistent with a previous report on fluorescence resonance energy transfer (FRET)-based RhoA indicators, which showed that indicators using an Rtkn-RBD had low signal contrast, whereas those using a PKN-derived RBD (PKN-RBD) showed high signal contrasts.³⁶ Therefore, we tested a version incorporating this PKN-derived RBD and observed a significantly improved signal contrast, with Q63L showing strong fluorescence and T19N minimal fluorescence (Figure S3C). Similar results were observed for the R-RhoA indicator (Figure S3D). Having confirmed the performance of our indicators, we then performed RGB multiplexed imaging using Lifeact-EBFP2, Gachapin, and the ddRFP-based Rho GTPase indicators. To perform the imaging, we established two types of stable HEK293A cell lines, namely cell A stably expressing Lifeact-EBFP2, Gachapin-A, and R-Rac1 or R-RhoA, and cell B stably expressing Gachapin-B (Figure 2B). Observation of co-cultured cells and line profiles showed that cell A surrounded by cell B separately exhibited fluorescent signals of Lifeact-EBFP2, Gachapin, and R-Rac1 or R-RhoA (Figures 2C, 2E, S4A, and S4C). We then performed time-lapse imaging of co-cultured cells. Kymographs along the lines (from Y to Y′) generated from time-lapse images over time showed that the dynamics of F-actin, cell-cell contact, and Rac1 or RhoA activities accompanied by cell movement were successfully monitored by Lifeact-EBFP2, Gachapin, and R-Rac1 or R-RhoA, respectively (Figures 2D, 2F, S4B, and S4D; Videos S3, S4, S5, S6, S7, and S8). These results demonstrate the availability of Gachapin for multiplexed functional live imaging.

Visualization of dynamic cell-cell contact using Gachapin

(A) Time-lapse images of cell-cell contact during cell movement using Gachapin. HEK293A cells individually expressing Gachapin components were co-cultured and observed every 3 min. Gachapin-A- and Gachapin-B-expressing cells were labeled by mCherry-NLS and EBFP2-NLS, respectively. Scale bars, 20 μm. The fluorescence intensity (F.I.) of Gachapin at the cell-cell contact sites indicated by arrowheads was plotted over time. Time-lapse imaging was performed at least three times. Other examples are included in Figure S1.

(B) Schematic of a co-culture experiment for visualizing F-actin, cell-cell contact, and Rho-family small GTPase activity. Two stable cell lines, namely cell A and cell B, were established. Cells A represent HEK293A cells stably expressing EBFP2-fused Lifeact (Lifeact-EBFP2), Gachapin-A, and a ddRFP-based Rho-family small GTPase activity indicator (R-Rac1 or R-RhoA). Cells B represent HEK293A cells stably expressing Gachapin-B.

(C) Simultaneous observation of F-actin, cell-cell contact, and Rac1 activity using Lifeact-EBFP2, Gachapin, and R-Rac1, respectively. Cells A and Cells B were co-cultured and observed using a confocal microscope. Cells B are indicated by yellow asterisks. Scale bars, 20 μm. Fluorescence profiles on the yellow line from X to X′ are shown at the bottom. Relative fluorescence intensities of Lifeact-EBFP2, Gachapin, and R-Rac1 are represented by blue, green, and red lines, respectively. Other examples are included in Figure S4A.

(D) Dynamics of F-actin, cell-cell contact, and Rac1 activity accompanied by cell movement. Kymographs showing changes in fluorescence intensities of the indicators accompanied by cell movement along the white line from Y to Y′ were generated from 240 time-lapse images acquired every 1 min. Scale bars, 20 μm. Time-lapse imaging was performed at least three times. Other examples are included in Figure S4B.

(E) Simultaneous observation of F-actin, cell-cell contact, and RhoA activity using Lifeact-EBFP2, Gachapin, and R-RhoA, respectively. The experimental and analytical conditions are the same as in (C). Other examples are included in Figure S4C.

(F) Dynamics of F-actin, cell-cell contact, and RhoA activity accompanied by cell movement. The experimental and analytical conditions are the same as in (D). Time-lapse imaging was performed at least three times. Other examples are included in Figure S4D.

See also Videos S1, S2, S3, S4, S5, S6, S7, and S8.

Video S1. Examples of cell-cell contact visualized by Gachapin and mGRASP, related to Figure 2

HEK293A cells individually expressing Gachapin components were co-cultured and imaged every 3 min. Gachapin-A- and Gachapin-B-expressing cells were labeled by mCherry-NLS and EBFP2-NLS, respectively. Scale bar, 20 μm.

Download video file^{(54.5KB, mp4)}

Video S2. Examples of cell-cell contact visualized by Gachapin and mGRASP, related to Figure 2

HEK293A cells individually expressing mGRASP components, pre-mGRASP and post-mGRASP, were co-cultured and imaged every 3 min. pre-mGRASP- and post-mGRASP-expressing cells were labeled by mCherry-NLS and EBFP2-NLS, respectively. Scale bar, 20 μm.

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Video S3. Examples of simultaneous observation of F-actin, cell-cell contact, and small GTPase activity using Lifeact-EBFP2, Gachapin, and a ddRFP-based Rho-family small GTPase activity indicator (R-Rac1 or R-RhoA), related to Figure 2

HEK293A cells stably expressing Lifeact-EBFP2, Gachapin-A, and R-Rac1 and those stably expressing Gachapin-B were co-cultured and imaged every 1 min. Scale bars, 20 μm.

Download video file^{(2.7MB, mp4)}

Video S4. Examples of simultaneous observation of F-actin, cell-cell contact, and small GTPase activity using Lifeact-EBFP2, Gachapin, and a ddRFP-based Rho-family small GTPase activity indicator (R-Rac1 or R-RhoA), related to Figure 2

HEK293A cells stably expressing Lifeact-EBFP2, Gachapin-A, and R-Rac1 and those stably expressing Gachapin-B were co-cultured and imaged every 1 min. Scale bars, 20 μm.

Download video file^{(2.3MB, mp4)}

Video S5. Examples of simultaneous observation of F-actin, cell-cell contact, and small GTPase activity using Lifeact-EBFP2, Gachapin, and a ddRFP-based Rho-family small GTPase activity indicator (R-Rac1 or R-RhoA), related to Figure 2

HEK293A cells stably expressing Lifeact-EBFP2, Gachapin-A, and R-Rac1 and those stably expressing Gachapin-B were co-cultured and imaged every 1 min. Scale bars, 20 μm.

Download video file^{(3.5MB, mp4)}

Video S6. Examples of simultaneous observation of F-actin, cell-cell contact, and small GTPase activity using Lifeact-EBFP2, Gachapin, and a ddRFP-based Rho-family small GTPase activity indicator (R-Rac1 or R-RhoA), related to Figure 2

HEK293A cells stably expressing Lifeact-EBFP2, Gachapin-A, and R-RhoA and those stably expressing Gachapin-B were co-cultured and imaged every 1 min. Scale bars, 20 μm.

Download video file^{(2.4MB, mp4)}

Video S7. Examples of simultaneous observation of F-actin, cell-cell contact, and small GTPase activity using Lifeact-EBFP2, Gachapin, and a ddRFP-based Rho-family small GTPase activity indicator (R-Rac1 or R-RhoA), related to Figure 2

HEK293A cells stably expressing Lifeact-EBFP2, Gachapin-A, and R-RhoA and those stably expressing Gachapin-B were co-cultured and imaged every 1 min. Scale bars, 20 μm.

Download video file^{(451KB, mp4)}

Video S8. Examples of simultaneous observation of F-actin, cell-cell contact, and small GTPase activity using Lifeact-EBFP2, Gachapin, and a ddRFP-based Rho-family small GTPase activity indicator (R-Rac1 or R-RhoA), related to Figure 2

HEK293A cells stably expressing Lifeact-EBFP2, Gachapin-A, and R-RhoA and those stably expressing Gachapin-B were co-cultured and imaged every 1 min. Scale bars, 20 μm.

Download video file^{(4.4MB, mp4)}

Application of Gachapin in neurons

We investigated the applicability of Gachapin to neurons. We individually expressed Gachapin components in dissociated hippocampal neurons and co-cultured them. Gachapin signal was detected at the contact sites of neuronal processes (Figures 3A, 3B, S5A, and S5B, line X–X′ in yellow rectangle). The signal was specifically detected between the neuronal processes of a Gachapin-A-expressing neuron labeled by mCherry and a Gachapin-B-expressing neuron labeled by EBFP2. In contrast, a process of a Gachapin-A-expressing neuron that was not in contact with a Gachapin-B-expressing neuron showed little fluorescence (Figures 3A, 3B, S5A, and S5B, line Y–Y′ in cyan rectangle). We next examined whether Gachapin visualizes the formation and disruption of process-process contact in neurons. Time-lapse imaging of co-cultured neurons individually expressing Gachapin components demonstrated that the dynamics of process-process contact in neurons are successfully monitored by Gachapin (Figures 3C and S5C).

Visualization of contact between different cells and between processes originating from a single cell

We have previously developed cIPAD, a fluorescent indicator for visualizing trans-interactions of clustered Pcdhα4 between different cells and between processes originating from a single cell.¹⁷^,¹⁸ To generalize it and visualize simple contact, rather than specific interactions of cell adhesion molecules, between different cells and between processes originating from a single cell, we set out to develop another indicator inspired by cIPAD. cIPAD was developed by inserting ddGFP-A and ddFP-B into EC1 and EC4 of Pcdhα4. Since EC1–EC4 mediate homophilic trans-interactions and EC5–EC6 mediate cis-interactions, using wild-type Pcdh could affect endogenous isoforms. To avoid this, we used chimeric Pcdh from PcdhγB2 and PcdhγA3, which interacts only with itself,²⁷ and employed EC1–EC5 to prevent cis-interactions with endogenous Pcdhs.²⁵^,²⁶ On the basis of these notions, we aimed to develop a Pcdh-based indicator capable of visualizing simple contact between processes originating from the same cell. We constructed a prototype indicator, named Gachapin-C1, which consists of ddGFP-A, ddFP-B3, a chimeric Pcdh, a 218 linker, and a GPI anchor (Figure 4A). We selected ddFP-B3 over ddFP-B1 because our previous report showed that cIPAD, a similar system, performed better with ddFP-B3.¹⁷ As an initial proof of concept, we tested if Gachapin-C1 could visualize contact between different cells. A Gachapin-C1 signal was indeed observed at the contact sites between two Gachapin-C1-expressing cells, which were marked by mCherry-NLS (Figure 4B). However, we found that Gachapin-C1 also emitted fluorescence at unexpected locations. The signal appeared not only at contact sites between two Gachapin-C1-expressing cells (Figure 4B, open arrowhead) but also, on occasion, at sites where a Gachapin-C1-expressing cell contacted a non-expressing cell (Figure 4B, closed arrowhead). Since the similarly designed cIPAD was able to specifically show fluorescence at the cell-cell contact sites in our previous work,¹⁷ we tried investigating whether the introduction of a cis-interacting motif, which is present in cIPAD but absent in Gachapin-C1, would allow us to specifically visualize cell-cell contact. We prepared Gachapin-C2 by introducing a parallel leucine zipper as a cis-interacting motif³⁷ between the 218 linker and the GPI anchor of Gachapin-C1 (Figure 4A). Gachapin-C2 fluorescence was mainly observed at cell-cell contact sites, with an intensity 2.02 times higher than Gachapin-C1 (Figures 4B and 4C). To precisely investigate the possibility that Gachapin-C1 and -C2 can emit fluorescence caused by intramolecular or intermolecular heterodimerization of ddGFP on the same membrane, we performed a co-culture experiment. We prepared two populations of cells: one expressing Gachapin-C1/Gachapin-C2 and mCherry-NLS (cell R), and the other expressing EBFP2-NLS (cell B) (Figure 4D). Co-culture experiments showed that Gachapin-C1 tended to emit fluorescence not only at R-R (Figure 4E, open arrowheads) but also at B-R (Figure 4E, closed arrowheads). In contrast, the Gachapin-C2 signal was specifically observed at R-R, yielding a higher signal contrast (8.10 vs. 2.92) (Figures 4E and 4F). These results indicate that Gachapin-C2 visualizes cell-cell contact with higher brightness. Hereafter, we used Gachapin-C2 as Gachapin-C. Cell aggregation assays confirmed that Gachapin-C expression did not induce cell adhesion, unlike PcdhγB2 (Figure S6A), and that Gachapin-C-expressing cells hardly co-aggregated with ancestral PcdhγA3- or PcdhγB2-expressing cells (Figure S6B). Next, we performed time-lapse imaging of serum-starved HEK293A cells expressing Gachapin-C to examine whether it can detect contact between processes extending from a single cell. The reason why we used serum-starved HEK293A cells is that we knew empirically that serum starvation causes HEK293A cells to extend their processes like neurons. We successfully monitored the formation and disruption of a contact site between processes extending from the same cell (Figures 4G and S6C, yellow arrowheads). These results show that Gachapin-C visualizes contact between different cells and between processes originating from a single cell, without inducing cell adhesion and without forming trans-interactions with ancestral Pcdh isoforms.

Development and characterization of Gachapin-C to visualize process-process contact originating from a single cell

(A) Schematics of two Gachapin-C variants. SS, GPI, 218, and LZ represent an acrosin-derived signal sequence, a Thy-1 N-terminal GPI-linked signal sequence, a flexible peptide linker whose sequence is GSTSGSGKPGSGEGSTKG, and a parallel leucine zipper, respectively.

(B) Visualization of cell-cell contact by Gachapin-C. HEK293T cells expressing Gachapin-C with mCherry-NLS were observed. Scale bars, 10 μm (left), 5 μm (right). Fluorescence profiles on the yellow line from X to X′ are shown on the right. Relative fluorescence intensity of Gachapin-C is represented by the green line. Cell-cell contact sites between Gachapin-C-expressing cells and those between a Gachapin-C-expressing cell and a non-expressing cell are indicated by open and closed arrowheads, respectively.

(C) Fluorescence intensities of Gachapin-C at the cell-cell contact sites were quantified. 505 (Gachapin-C1) and 430 (Gachapin-C2) cell-cell contact sites from six independent cultures were analyzed. The plot shows data from all individual cell-cell contact sites (gray dots). The mean value from each independent culture is overlaid as a red dot. The final value is presented as the mean ± SD of these independent culture means. Statistical significance was assessed by unpaired t test using mean values of six independent cultures.

(D) Schematics of the co-culture experiment between cells with or without expression of Gachapin-C variants. Cells with and without Gachapin-C variant expression were labeled by mCherry-NLS and EBFP2-NLS, respectively.

(E) HEK293T cells expressing the indicated constructs were co-cultured and observed using a confocal microscope. Cell-cell contact sites between mCherry-positive cells (red cells) and those between EBFP2-positive cells (blue cells) and red cells are denoted as R-R and B-R, respectively. Closed and open arrowheads indicate cell-cell contact sites formed by red cells/blue cells (B-R) and red cells/red cells (R-R), respectively. Scale bars, 10 μm.

(F) Fluorescence intensities of Gachapin-C variants at the R-R and B-R cell-cell contact sites were quantified. 114 (R-R, Gachapin-C1), 92 (B-R, Gachapin-C1), 104 (R-R, Gachapin-C2), and 80 (B-R, Gachapin-C2) cell-cell contact sites from four independent cultures were analyzed. The plot shows data from all individual cell-cell contact sites (gray dots). The mean value from each independent culture is overlaid as a red dot. The final value is presented as the mean ± SD of these independent culture means.

(G) Time-lapse images of process-process contact originating from a single cell using Gachapin-C. HEK293A cells expressing Gachapin-C and mCherry-NLS were serum-starved and observed every 3 min. A process-process contact site originating from a single cell is indicated by a yellow arrowhead. Scale bars, 10 μm. Representative images from at least two independent experiments are shown. Another example is included in Figure S6C.

Application of Gachapin-C in neurons

We next examined the applicability of Gachapin-C in neurons. The Gachapin-C signal was observed at the contact sites between cells but not at free processes (Figures 5A, 5B, S7A, and S7B). Gachapin-C also emitted fluorescence at process-process contact regions on the same neuron but not at free processes (Figures 5C, 5D, S7C, and S7D). We finally examined the dynamics of contact between processes originating from a single neuron. We observed dissociated hippocampal neurons expressing Gachapin-C (Figure 5E). Time-lapse imaging of the contact visualized by Gachapin-C on processes at the white square region in Figure 5E was performed. We successfully observed the formation and disruption of process-process contact (Figures 5F, S7E, and S7F, yellow arrowheads). These results suggest that Gachapin-C is applicable to neurons and can visualize contact not only between neurons but also between processes originating from a single neuron.

Discussion

Our development of Gachapin and Gachapin-C provides a versatile platform for visualizing dynamic cell-cell contacts, including interactions between distinct cells as well as between processes of a single cell. These fluorescent indicators leverage a reversible ddGFP approach to overcome the limitations of classical split-GFP-based contact indicators. Gachapin, comprising two complementary membrane-anchored components (Figure 1A), robustly labels cell contact sites without artificially promoting adhesion (Figures 1B–1E), confirming that our design visualizes contacts per se rather than inducing them. These results support our hypothesis that membrane-targeted ddGFP halves can report cell contacts in real time without perturbing normal cell-cell adhesion, a critical requirement for faithful imaging of dynamic interactions.

A major finding is that Gachapin can capture the formation and disruption of cell contacts in real time, unlike split-GFP systems. Time-lapse imaging in motile cells showed Gachapin fluorescence appearing when two cells establish contact and disappearing upon their detachment (Figures 2A and S1; Video S1). In contrast, mGRASP, a split-GFP indicator, failed to track contact disassembly under the same conditions (Figure S2; Video S2). This difference is attributable to the irreversible reconstitution and slow chromophore maturation of split-GFP, versus the rapid, reversible association of ddGFP. Thus, Gachapin uniquely enables monitoring of transient, dynamic cell-cell interactions that were previously invisible to GRASP-like indicators. In support of this advantage, our multiplexed imaging demonstrated that Gachapin can be combined with other fluorescent indicators to correlate structural contact events with cellular signaling dynamics. By co-expressing blue Lifeact-EBFP2 (reporting F-actin) and red ddRFP-based indicators for Rac1 or RhoA activity alongside green Gachapin, we simultaneously visualized contact formation, cytoskeletal remodeling, and Rho GTPase signaling in migrating cells (Figures 2B–2F and S4; Videos S3, S4, S5, S6, S7, and S8). This multiplex functional imaging underscores the utility of Gachapin for dissecting contact-mediated signaling events in live cells.

Extending our analysis to neurons, we found that Gachapin effectively labels contacts between neuronal processes. When hippocampal neurons were transfected such that one neuron expressed Gachapin-A and another expressed Gachapin-B, fluorescent signals arose specifically at points of contact between their neurites (Figures 3A, 3B, S5A, and S5B). No fluorescence was seen in processes that lacked a contacting partner, confirming the contact dependence of signal. Importantly, Gachapin could also visualize the dynamics of neurite-neurite contacts: time-lapse imaging captured neurites forming transient contacts and then retracting (Figures 3C and S5C), indicating that neuronal process interactions can be monitored in real time. These data suggest that Gachapin is suitable for studying early-stage neuronal circuit formation, where axons and dendrites explore and make/break physical contacts before synapses mature. Notably, our experiments were performed at 2–3 days in vitro (DIV 2–3), when synapses are not fully formed, so Gachapin’s signals in neurons likely reflect nascent or non-synaptic contacts. We did not observe clear synaptic puncta labeling at this stage, and whether Gachapin can visualize mature synapses remains uncertain. Nevertheless, the ability to observe neuronal processes touch and withdraw provides a window into phenomena such as early synaptogenesis.

An innovation of our study is the creation of Gachapin-C, a single-component contact indicator that can be expressed in cells of interest to report both intercellular contacts and self-contacts within the same cell. Gachapin-C was inspired by our previous cIPAD design for Pcdh interactions,¹⁷^,¹⁸ but here we generalized it to detect any membrane contacts by embedding ddGFP-A and ddFP-B within a chimeric Pcdh extracellular domain on one polypeptide. The initial prototype, Gachapin-C1, confirmed that one-component detection is possible: when two Gachapin-C1-expressing cells contacted each other, a fluorescent junction formed (Figure 4B). However, we also found a key challenge: Gachapin-C1 sometimes fluoresced on cell surfaces even in the absence of contact with other Gachapin-C1-expressing cells (Figure 4B, closed arrowhead), implying that ddGFP-A and ddFP-B within the same membrane were interacting in cis or folding intramolecularly to reconstitute fluorescence. This unintended leak signal was problematic. To suppress this, we introduced a parallel leucine zipper motif into the Gachapin-C1 construct (Gachapin-C2) to promote cis-dimerization of the indicators on the same cell membrane. This strategy was inspired by natural cell adhesion molecules (like Pcdhs) that use cis-interactions to organize and restrict their binding sites. The improved Gachapin-C2 showed markedly reduced leak signal: its fluorescence was almost exclusively observed at contacts between two Gachapin-C2-expressing cells and not between an expressing and a non-expressing cell (Figures 4E and 4F). Quantitatively, Gachapin-C2’s signal at cell-cell contact was ∼8-fold higher than any background, whereas the original Gachapin-C1 had a poorer ∼3-fold contrast. Gachapin-C2 also produced ∼2 times brighter junction signals than Gachapin-C1 (Figure 4C). We confirmed that Gachapin-C2 does not induce cellular adhesion or aggregate formation on its own: K562 cell aggregation assays showed no difference from a control, and Gachapin-C did not cross-interact with wild-type Pcdhs from the same family (Figures S6A and S6B). Therefore, the final Gachapin-C (corresponding to Gachapin-C2) provides a reliable one-component indicator of contact, suitable for cases where introducing two separate constructs is impractical. The incorporation of a cis-dimerization motif was key to its performance, although future work is needed to directly verify that the leucine zipper mediates cis-interactions as designed. It is intriguing to consider that this engineered solution may mirror a mechanism used by real cells: Pcdhs, for example, form cis-dimers that could similarly prevent unwanted self-binding. Investigating whether native adhesion molecules employ such cis-clustering to avert nonspecific interactions could yield biological insight.

In neuronal cultures, Gachapin-C proved capable of visualizing both inter-neuronal contacts and contacts between processes of the same neuron. When neurons in a dish expressed Gachapin-C, fluorescent puncta appeared where two neurons touched (Figures 5A, 5B, S7A, and S7B) and were absent on solitary neurites, confirming specificity to contact sites. Remarkably, we also observed Gachapin-C fluorescence where branches of the same neuron encountered each other (Figures 5C, 5D, S7C, and S7D). Time-lapse imaging of a single neuron’s processes revealed transient flashes of Gachapin-C signal as a process contacted another process and then withdrew (Figures 5E, 5F, S7E, and S7F). Such events are thought to be common during neural development—for example, dendrites of the same neuron often approach each other and then repel in a self-avoidance process that ensures proper tiling of receptive fields. In particular, cerebellar Purkinje cells and retinal starburst amacrine cells exhibit dendritic self-avoidance by transient self-contacts,³⁸^,³⁹^,⁴⁰^,⁴¹ and Gachapin-C now offers a means to monitor these interactions in real time. Another interesting case is the formation of autapses, where a neuron forms synaptic contacts onto itself. Autaptic self-connections have been documented in certain cortical pyramidal neurons and interneurons.⁴²^,⁴³^,⁴⁴ While our culture conditions were not optimized to form mature autapses, it is conceivable that expressing Gachapin-C in neurons could help detect autaptic contacts as they form. We expect that applying Gachapin-C in more mature neuronal networks or brain slice preparations will illuminate whether and how often neurons establish fleeting self-connections before stabilizing their wiring. Taken together, the Gachapin-C results establish that one-component contact indicators can reveal complex patterns of connectivity, including contacts within a single cell that are essential for self/non-self-discrimination in neural circuit assembly.

Limitations of the study

Gachapin and Gachapin-C have some limitations that warrant discussion. First, as noted above, we have not yet demonstrated these indicators in mature synapses or in intact tissue. Our assays used immature neuronal cultures where contacts are largely non-synaptic. It remains unclear if Gachapin signals would effectively mark bona fide synapses, which are enriched in specialized adhesion molecules and have tight junctional clefts. It is possible that in a mature synapse, the intermembrane gap or molecular crowding could affect the heterodimerization of ddGFP. Additionally, because Gachapin is a general contact indicator, it will label all points of membrane contact, not distinguishing synaptic versus non-synaptic contacts. In a dense neural tissue, this could lead to pervasive fluorescence, making it challenging to isolate specific synaptic connections. For studies specifically targeting synapses, a targeted indicator like SynapShot¹⁹ may provide higher specificity. The choice of tool will depend on the biological question. A logical future step will be to test Gachapin and Gachapin-C in more developed neural networks (e.g., later DIV or organotypic slices) to see if they can highlight synaptic contacts or if their use should be confined to earlier developmental stages and non-synaptic interactions. Encouragingly, the success of SynapShot in vivo suggests that ddFP-based indicators are compatible with the in vivo brain environment; thus, Gachapin should also be translatable with appropriate delivery and imaging methods.

Another limitation involves potential background and sensitivity issues. Although Gachapin-C2 greatly reduced the leakage problem of the one-component design, background fluorescence independent of cell-cell contact could occur when expression levels are very high. Complete elimination of such false signals might require further protein engineering or using additional strategies (for example, inducible or low-level expression to minimize the chance of self-activation). In our hands, Gachapin-C’s signal-to-noise ratio was robust (Figure 4F), but careful controls (like the mixed culture assay in Figure 4E) should be employed in new cell types to ensure specificity.

We also acknowledge that overexpression of these indicators could have subtle effects on cells. We did not observe overt changes in toxicity—for instance, neurite outgrowth in neurons was not detectably altered by expressing Gachapin or Gachapin-C (Figures S7G–S7I)—and the indicators themselves did not trigger adhesion (Figures 1E, S6A, and S6B). However, we did not rigorously assess every aspect of cell physiology. It remains possible that, in some contexts, the presence of membrane-tethered ddGFP fragments could influence membrane properties or interact with endogenous proteins. Any such effects are likely minor (as suggested by our controls), but future users of the system should remain vigilant and include appropriate controls (e.g., cells expressing only one component, or a non-functional mutant indicator) to ensure that the observed biological effects truly stem from natural contact events and not from the indicator itself.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Takashi Kanadome (kanadome@nibb.ac.jp).

Materials availability

Plasmids generated in this study are available from the lead contact on reasonable request.

Data and code availability

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Data reported in this paper will be shared by the lead contact upon request.
•
This paper does not report original code.
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Any additional information required for data reported in this paper is available from the lead contact upon reasonable request.

Acknowledgments

We thank the Optics and Imaging Faculty of the National Institute for Basic Biology for the use of their confocal microscope. This work was supported by the JST PRESTO Program (nos. JPMJPR2045 to T.K. and JPMJPR2149 to H.H.), the MEXT Grant-in-Aid for Scientific Research on Transformative Research Areas (A) (nos. JP23H04703 to H.H. and JP22H05498 to T.Y.), JSPS Grant-in-Aid for Scientific Research (A) (no. JP18H04016 to T.Y.), JSPS Grant-in-Aid for Scientific Research (B) (no. JP24K02781 to S.J.), Narishige Neuroscience Research Foundation to T.K., and Nakatani Foundation for Advancement of Measuring Technologies in Biomedical Engineering to T.K.

Author contributions

Conceptualization, T.K.; methodology, T.K.; formal analysis, T.K.; investigation, T.K., N.H., and H.H.; writing – original draft, T.K.; writing – review and editing, T.K., N.H., S.J., H.H., T.Y., and T.N.; supervision, T.K., T.Y., and T.N.; project administration, T.K.; funding acquisition, T.K., S.J., H.H., and T.Y.

Declaration of interests

The authors declare no competing interests.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work, the authors used ChatGPT and Gemini for manuscript proofreading. After using these tools, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Bacterial and virus strains

Escherichia coli strain XL10-Gold	Agilent Technologies	Cat# 200314

Chemicals, peptides, and recombinant proteins

NheI	Takara	Cat# 1241A
KpnI	Takara	Cat# 1068AH
BspEI	New England Biolabs	Cat# R0540S
BglII	Takara	Cat# 1021A
MluI	Takara	Cat# 1071A
EcoRI	Takara	Cat# 1040A
NotI	Takara	Cat# 1166A
SphI	Takara	Cat# 1246A
Dulbecco’s modified Eagle’s medium (Low Glucose) with L-Glutamine and Phenol Red	Fujifilm	Cat# 041-29775
Fetal bovine serum	Biowest	N/A
Fetal bovine serum	Thermo Fisher Scientific	N/A
Fetal bovine serum	Biosera	Cat# 55633865 Lot# S000P
Iscove’s modified Dulbecco’s medium	Thermo Fisher Scientific	Cat# 12440053
Polyethylenimine “MAX”	Cosmo Bio	Cat# 24765-1
Neuron Dissociation Solutions	Wako	Cat# 291-78001
Minimum essential media	Thermo Fisher Scientific	Cat# 11090-081
FluoroBrite DMEM	Thermo Fisher Scientific	Cat# A1896701
B-27 supplement	Thermo Fisher Scientific	Cat# 17504044
GlutaMAX	Thermo Fisher Scientific	Cat# 35050061
Penicillin-Streptomycin (10,000 U/mL)	Thermo Fisher Scientific	Cat# 15140122
HEPES (1 M)	Thermo Fisher Scientific	Cat# 15630080
Poly-L-lysine hydrobromide	Sigma-Aldrich	Cat# P2636
Cytosine β-D-arabinofuranoside hydrochloride	Sigma-Aldrich	Cat# C6645
Trypsin-EDTA (0.25%) and phenol red	Thermo Fisher Scientific	Cat# 25200072
DMEM/F12, HEPES, no phenol red	Thermo Fisher Scientific	Cat# 11039-021
Cellmatrix Type I-C	Nitta gelatin	Cat# 631-00771
Polyethylenimine	Sigma-Aldrich	Cat# P3143
IMDM, no phenol red	Thermo Fisher Scientific	Cat# 21056023
Puromycin	InvivoGen	Cat# ant-pr-1
Blasticidin	InvivoGen	Cat# ant-bl-1

Experimental models: Cell lines

HEK293T	RIKEN BRC	N/A
K562	RIKEN BRC	N/A
HEK293A	Thermo Fisher Scientific	Cat#R705-07
HeLa	RIKEN BRC	N/A

Experimental models: Organisms/strains

Mouse: C57/B6J	Japan SLC	N/A

Recombinant DNA

pCX:Gachapin-1A-P2A-mCherry-NLS	This paper	N/A
pCX:Gachapin-1B-P2A-EBFP2-NLS	This paper	N/A
pCX:Gachapin-2A-P2A-mCherry-NLS	This paper	N/A
pCX:Gachapin-2B-P2A-EBFP2-NLS	This paper	N/A
pCX:Gachapin-3A-P2A-mCherry-NLS	This paper	N/A
pCX:Gachapin-3B-P2A-EBFP2-NLS	This paper	N/A
pCX:Gachapin-4A-P2A-mCherry-NLS (pCX:Gachapin-A-P2A-mCherry-NLS)	This paper	Addgene 251133
pCX:Gachapin-4B-P2A-EBFP2-NLS (pCAG:Gachapin-B-P2A-EBFP2-NLS)	This paper	Addgene 251134
pCAGGS:mCherry-NLS	Kanadome et al.¹⁶	N/A
pCAGGS:EBFP2-NLS	Kanadome et al.¹⁶	N/A
pCAGGS:NCad	Kanadome et al.¹⁶	N/A
pCAGGS:pre-mGRASP-P2A-EBFP2-NLS	Kanadome et al.¹⁶	N/A
pCAGGS:post-mGRASP-P2A-mCherry-NLS	Kanadome et al.¹⁶	N/A
pCAGGS:G-RhoA Q63L (PKN-RBD)	This paper	N/A
pCAGGS:G-RhoA T19N (PKN-RBD)	This paper	N/A
pCAGGS:G-RhoA Q63L (Rtkn-RBD)	This paper	N/A
pCAGGS:G-RhoA T19N (Rtkn-RBD)	This paper	N/A
pCAGGS:R-RhoA Q63L (PKN-RBD)	This paper	N/A
pCAGGS:R-RhoA T19N (PKN-RBD)	This paper	N/A
pCAGGS:miRFP670	This study	N/A
pPBbsr2:Gachapin-A-P2A-Lifeact-EBFP2	This study	N/A
pPBbsr2:Gachapin-B	This study	N/A
pBRPB-CAG:R-Rac1	This study	N/A
pBRPB-CAG:R-RhoA	This study	N/A
Super PiggyBac Transposase Expression Vector	System Biosciences	Cat#PB210PA-1
pCX:Gachapin-A-P2A-mCherry	This study	Addgene 251135
pCX:Gachapin-B-P2A-EBFP2	This study	Addgene 251136
pCX:Gachapin-C1-P2A-mCherry-NLS	This study	N/A
pCX:Gachapin-C2-P2A-mCherry-NLS (pCX:Gachapin-C-P2A-mCherry-NLS)	This study	Addgene 251137
pCX:Gachapin-C-P2A-mCherry	This study	Addgene 251138
pCX:PcdhγB2ΔICD-Venus	Kanadome et al.²⁷	N/A
pCX:PcdhγB2ΔICD-EBFP2	Kanadome et al.¹⁷	N/A
pCX:PcdhγA3ΔICD-EBFP2	Kanadome et al.¹⁷	N/A

Software and algorithms

GraphPad Prism9	GraphPad Software, Inc.	https://www.graphpad.com/ RRID: SCR_002798
ImageJ (FIJI)	Schneider et al.⁴⁵	https://imagej.nih.gov/ij/ RRID: SCR_002285
Imaris	Oxford Instruments	https://imaris.oxinst.com/ RRID: SCR_007370

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Experimental model and study participant details

Cell culture

HEK293T (RIKEN BRC), HEK293A (Thermo Fisher Scientific), HeLa (RIKEN BRC) cells, and HEK293A stable cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Fujifilm) supplemented with 10% (v/v) fetal bovine serum (FBS, Biowest or Biosera) at 37°C in humidified air containing 5% CO₂. K562 cells (RIKEN BRC) were maintained in Iscove’s modified Dulbecco’s medium (IMDM, Thermo Fisher Scientific) supplemented with 10% (v/v) FBS. All cell lines used in this study (HEK293T, HEK293A, HeLa, and K562) are of female origin.

For primary culture of hippocampal neurons, a pregnant mouse was anesthetized with CO₂ and sacrificed by cervical dislocation. The hippocampi were dissected under a microscope. Hippocampi were collected from E16–18 embryos (C57BL/6J) of either sex and were digested with Neuron Dissociation Solutions (Wako) according to the manufacturer’s protocol. Minimum Essential Media (MEM, Thermo Fisher Scientific) or FluoroBrite DMEM (Thermo Fisher Scientific) were supplemented with 5.5% FBS (Thermo Fisher Scientific), 2% B27 supplement (Thermo Fisher Scientific), 1 mM GlutaMAX (Thermo Fisher Scientific), 100 units/mL Penicillin-100 μg/mL streptomycin (Thermo Fisher Scientific), and 10 mM HEPES (1 M) (Gibco). The cells were suspended in a MEM-based medium and plated on a poly-L-lysine (Sigma-Aldrich) treated CELLview glass bottom dish (Advanced TC, 4-compartments) (Greiner). After 2 h, the medium was replaced with the FluoroBrite DMEM-based medium. The following day, 5 mM cytosine β-D-arabinofuranoside hydrochloride (Sigma-Aldrich) was added to a final concentration of 5 nM. The cells were incubated at 37°C in humidified air containing 5% CO₂, without any medium changes.

Animals

Animal experimentation was performed according to the Institutional Guidelines on Animal Experimentation at The University of Osaka (approval number: FBS-22-008). Pregnant female C57BL/6J mice were purchased from Japan SLC. The pregnant mice were single housed for a few days in a temperature-controlled (22°C) room with a 12-h light/dark cycle with free access to food and water. E16–18 embryos of either sex were used for primary culture of hippocampal neurons. Typically, two embryos were used for each experiment.

Method details

Plasmid construction

pCX:Gachapin-1A-P2A-mCherry-NLS and pCX:Gachapin-1B-P2A-EBFP2-NLS were constructed by insertion of ddGFP-A- and ddFP-B1-coding sequences into NheI/KpnI sites of pCX:ss-NheI-KpnI-GPI-P2A-mCherry-NLS and pCX:ss-NheI-KpnI-GPI-P2A-EBFP2-NLS vectors, respectively, which had been constructed by overlapping PCR from pCAG:GPI-GFP (Addgene plasmid #32601)⁴⁶ and mCherry-NLS- or EBFP2-NLS-coding sequences, respectively. pCX:Gachapin-2A-P2A-mCherry-NLS and pCX:Gachapin-2B-P2A-EBFP2-NLS were constructed by insertion of a hybridized oligo coding a 218 linker into a BspEI site of pCX:Gachapin-1A-mCherry-NLS and pCX:Gachapin-1B-P2A-EBFP2-NLS, respectively. pCX:Gachapin-3A-P2A-mCherry-NLS and pCX:Gachapin-3B-P2A-EBFP2-NLS were constructed by insertion of ddGFP-A- and ddFP-B1-coding sequences into NheI-digested pCX:Gachapin-1A-P2A-mCherry-NLS and pCX:Gachapin-1B-P2A-EBFP2-NLS, respectively. pCX:Gachapin-4A-P2A-mCherry-NLS and pCX:Gachapin-4B-P2A-EBFP2-NLS were constructed by insertion of a hybridized oligo coding a 218 linker into a BspEI site of pCX:Gachapin-3A-P2A-mCherry-NLS and pCX:Gachapin-3B-P2A-EBFP2-NLS, respectively. pCX:Gachapin-A-P2A-mCherry and pCX:Gachapin-B-P2A-EBFP2 were constructed by replacing mCherry-NLS- and EBFP2-NLS- coding sequences flanked by BspEI and BglII sites of pCX:Gachapin-A-P2A-mCherry-NLS and pCX:Gachapin-B-P2A-EBFP2-NLS with mCherry- and EBFP2-coding sequences, respectively. To generate mutant pCAGGS:G-RhoAs (PKN-RBD), wild-type pCAGGS:G-RhoA (PKN-RBD) was first constructed by overlapping PCR to assemble ddFP-B3-, PKN-RBD (human PKN, 13–98 a.a.)-, P2A-, ddGFP-A-, and RhoA (human RhoA 1–193 a.a.)-coding sequences, followed by subcloning into a pCAGGS vector. PKN-RBD- and RhoA-coding sequences were obtained from pCAGGS-Raichu-RhoA-CR (Addgene plasmid #40258).⁴⁷ Since pCAGGS-Raichu-RhoA-CR contains only a part of RhoA (human RhoA 1–189 a.a.), 190–193 a.a. of RhoA was added by PCR using a reverse primer containing the corresponding sequence. Mutant pCAGGS:G-RhoAs (PKN-RBD) were constructed by site-directed mutagenesis of wild-type pCAGGS:G-RhoA (PKN-RBD). To create mutant pCAGGS:G-RhoAs (Rtkn-RBD), the PKN-RBD-coding sequence flanked by KpnI and MluI sites of mutant pCAGGS:G-RhoAs (PKN-RBD) was replaced by a synthesized gene fragment coding Rtkn-RBD (mouse Rhotekin isoform 2, 1–88 a.a.) (Integrated DNA Technologies). Mutant pCAGGS:R-RhoAs (PKN-RBD) were constructed by replacing the ddGFP-A-coding sequence flanked by MluI and BspEI sites of mutant pCAGGS:G-RhoAs (PKN-RBD) with the ddRFP-A-coding sequence. pCAGGS:miRFP670 was prepared by cloning of the miRFP670-coding sequence into EcoRI/NotI sites of a pCAGGS vector. For construction of pPBbsr2:Gachapin-A-P2A-Lifeact-EBFP2, overlapping PCR was performed to assemble Gachapin-A-, P2A-, and Lifeact-EBFP2-coding sequences, and then subcloned into EcoRI/BglII sites of a pPBbsr2 vector prepared from pPBbsr2-4031-NES (Addgene plasmid #105241).⁴⁸ pPBbsr2:Gachapin-B was constructed by insertion of the Gachapin-B-coding sequence into EcoRI/BglII sites of a pPBbsr2 vector. To create pBRPB-CAG:R-Rac1, pCAGGS:G-Rac1 was first constructed by replacing the PKN-RBD-coding sequence flanked by KpnI/MluI sites and the RhoA-coding sequence flanked by BspEI/NotI sites with synthesized gene fragments coding PAK1CRIB (human PAK1, 68–150 a.a.) and Rac1 (human Rac1, 1–192 a.a.) (Integrated DNA Technologies), respectively. Next, pCAGGS:R-Rac1 was constructed by replacing the ddGFP-A-coding sequence flanked by MluI and BspEI sites of pCAGGS:G-Rac1 with the ddRFP-A-coding sequence. Finally, pBRPB-CAG:R-Rac1 was constructed by insertion of the R-Rac1-coding sequence into a pBRPB-CAG vector prepared by pBRPB CAG-mCherry-IP (Addgene plasmid #106333).⁴⁹ pBRPB-CAG:R-RhoA was constructed by cloning of the R-RhoA-coding sequence into a pBRPB-CAG vector. pCX:Gachapin-C1-P2A-mCherry-NLS was constructed by inserting the γB2EC1-γA3EC2–EC3-γB2EC4-coding sequence flanked by AgeI/MluI sites and the γB2EC4–EC5 coding sequence flanked by KpnI sites amplified by PCR from pCX:γB2γA3ΔICD-EC5-VenusΔN3C9-P3²⁷ and pCX:PcdhγB2, respectively, into a pCX:ss-ddFP-B3-AgeI-MluI-ddGFP-A-KpnI-218-P2A-mCherry-NLS prepared in advance. pCX:Gachapin-C2-P2A-mCherry-NLS was constructed by inserting a synthesized gene fragment coding a parallel leucine zipper³⁷ (Integrated DNA Technologies) into a BspEI site of pCX:Gachapin-C1-P2A-mCherry-NLS. pCX:Gachapin-C-P2A-mCherry was constructed by replacing the γB2EC5-GPI-P2A-mCherry-NLS-coding sequence flanked by SphI and BglII sites with the γB2EC5-GPI-P2A-mCherry-coding sequence.

Plasmid transfection

HEK293T, HEK293A, and HeLa cells were transfected using polyethylenimine MAX (Cosmo Bio). K562 cells were electroporated using an electroporator (NEPA21: NEPAGENE) with the following conditions: 1 × 10⁶ dissociated cells, 10 μg of plasmid, poring pulse (275 V, 1 ms pulse length, 50 ms interval, twice, 10% decay rate), and transfer pulse (20 V, 50 ms pulse length, 50 ms interval, 5 times, 40% decay rate). Dissociated neurons were electroporated using an Amaxa 4D-Nucleofector (Lonza).

Establishment of HEK293A stable cell lines

HEK293A stably expressing Lifeact-EBFP2, Gachapin-A, and ddRFP-based Rho-family small GTPase activity indicators (R-Rac1 or R-RhoA) were established by co-transfection of Super PiggyBac Transposase Expression Vector (System Biosciences), pPBbsr2:Gachapin-A-P2A-Lifeact-EBFP2, and pBRPB-CAG:R-Rac1 or pBRPB-CAG:R-RhoA, followed by selection using 1 μg/mL of puromycin (InvivoGen) and 10 μg/mL of blasticidin (InvivoGen). HEK293A stably expressing Gachapin-B were established by co-transfection of Super PiggyBac Transposase Expression Vector and pPBbsr2:Gachapin-B, followed by selection using 10 μg/mL of blasticidin.

Cell imaging

HEK293T, HEK293A, and HeLa cells grown on glass-bottomed dishes coated with Cellmatrix Type I-C (Nitta gelatin) were transiently transfected with expression plasmids and incubated overnight. Before imaging, the medium was replaced with phenol red-free DMEM/F12 (Thermo Fisher Scientific). Fluorescence and DIC images of HEK293T and HEK293A cells were acquired using an Olympus FV-1000 laser scanning confocal microscope with an IX81 microscope equipped with a ×60, 1.35 numerical aperture (NA) oil-immersion objective lens (UPLSAPO60XO) (Olympus). The excitation wavelengths for EBFP2, ddGFP, mCherry, ddRFP, and miRFP670 were 405, 488, 543, 543, and 633 nm, respectively. For co-culture experiments, HEK293T and HEK293A cells were individually transfected with expression plasmids and incubated overnight. The cells were detached from the dishes using trypsin-EDTA (0.25%) and phenol red (Thermo Fisher Scientific), mixed in 10% FBS/DMEM, and seeded on glass-bottomed dishes coated with Cellmatrix Type I-C. Imaging was performed after 24 h. Time-lapse imaging of HEK293A cells grown in phenol red-free DMEM/F12 containing 10% FBS and 100 U/mL Penicillin-Streptomycin (Thermo Fisher Scientific) was performed using an Olympus FV-1000 laser scanning confocal microscope with an IX81 microscope equipped with a ×40, 1.35 NA oil-immersion objective lens (UApo/340 40x/1.35) (Olympus) and a stage incubator heated at 37°C. K562 cells were observed using an Olympus FV-1000 laser scanning confocal microscope with an IX81 microscope equipped with ×10, 0.40 NA objective lens (UPlanApo 10×/0.40) (Olympus). Fluorescence images of HeLa cells were acquired using an Olympus FV-1200 laser scanning confocal microscope with an IX83 microscope equipped with a ×60, 1.35 NA oil-immersion objective lens (UPlanSApo 60×/1.35) (Olympus). The excitation wavelengths for ddGFP, ddRFP, and miRFP670 were 473, 559, and 635 nm, respectively. Primary cultures of hippocampal neurons were observed at days in vitro (DIV) 2–3. Fluorescence images were acquired using an LSM900 laser scanning confocal microscope with a 63x, 1.4 NA oil-immersion objective lens (Plan-Apochromat 63×/1.40 Oil DIC M27) (Zeiss). The excitation wavelengths for EBFP2, ddGFP, and mCherry were 405, 488, and 561 nm, respectively. Image analysis was performed using ImageJ (FIJI)⁴⁵ to quantify fluorescence intensities and generate Plot profiles, Lookup Tables (LUT), and Kymographs. In neuronal cultures, total neurite length, the number of primary neurites emerging from the soma, and the number of branch points per neuron were quantified using the Filament Tracer module in Imaris (version 9.3.1; Oxford Instruments).

Cell aggregation assay

Cell aggregation assay using K562 cells was performed as described previously.¹⁶^,¹⁷ Transfected K562 cells were cultured in IMDM, no phenol red (Thermo Fisher Scientific) supplemented with 10% FBS and 100 U/mL Penicillin-Streptomycin under rotation at 30 rpm overnight at 37°C in humidified air containing 5% CO₂. Before imaging, cells were transferred by decantation to glass-bottomed dishes coated with 0.1% (w/v) polyethylenimine (P3143, Sigma-Aldrich) and incubated for 1 h at 37°C in humidified air containing 5% CO₂. CoAg Index was calculated based on a previous report.³⁰

Quantification and statistical analysis

Statistical analysis was performed using GraphPad Prism 9 (GraphPad Software, Inc.). Ordinary one-way ANOVA, followed by Tukey’s multiple comparison test was performed for the analysis in Figures 1C, 1E, and S7G–S7I. Unpaired t test was performed for the analysis in Figures 4C and S3B–S3D. p values < 0.05 were considered statistically significant. Data in Figures 1C, 1E, 4C, 4F, S3B–S3D, S6B, and S7G–S7I are shown as the mean ± SD. Sample sizes are listed in the Figure legends for each experiment. Reproducibility was confirmed by at least three independent experiments, with the exception of Figures 4G and 5F, which were confirmed by at least two independent experiments.

Published: January 23, 2026

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.crmeth.2025.101292.

Supplemental information

Document S1. Figures S1–S7

mmc1.pdf^{(20MB, pdf)}

Document S2. Article plus supplemental information

mmc10.pdf^{(39.4MB, pdf)}

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Video S1. Examples of cell-cell contact visualized by Gachapin and mGRASP, related to Figure 2

Download video file^{(54.5KB, mp4)}

Video S2. Examples of cell-cell contact visualized by Gachapin and mGRASP, related to Figure 2

Download video file^{(57.3KB, mp4)}

HEK293A cells stably expressing Lifeact-EBFP2, Gachapin-A, and R-Rac1 and those stably expressing Gachapin-B were co-cultured and imaged every 1 min. Scale bars, 20 μm.

Download video file^{(2.7MB, mp4)}

HEK293A cells stably expressing Lifeact-EBFP2, Gachapin-A, and R-Rac1 and those stably expressing Gachapin-B were co-cultured and imaged every 1 min. Scale bars, 20 μm.

Download video file^{(2.3MB, mp4)}

HEK293A cells stably expressing Lifeact-EBFP2, Gachapin-A, and R-Rac1 and those stably expressing Gachapin-B were co-cultured and imaged every 1 min. Scale bars, 20 μm.

Download video file^{(3.5MB, mp4)}

HEK293A cells stably expressing Lifeact-EBFP2, Gachapin-A, and R-RhoA and those stably expressing Gachapin-B were co-cultured and imaged every 1 min. Scale bars, 20 μm.

Download video file^{(2.4MB, mp4)}

HEK293A cells stably expressing Lifeact-EBFP2, Gachapin-A, and R-RhoA and those stably expressing Gachapin-B were co-cultured and imaged every 1 min. Scale bars, 20 μm.

Download video file^{(451KB, mp4)}

HEK293A cells stably expressing Lifeact-EBFP2, Gachapin-A, and R-RhoA and those stably expressing Gachapin-B were co-cultured and imaged every 1 min. Scale bars, 20 μm.

Download video file^{(4.4MB, mp4)}

Document S1. Figures S1–S7

mmc1.pdf^{(20MB, pdf)}

Document S2. Article plus supplemental information

mmc10.pdf^{(39.4MB, pdf)}

Data Availability Statement

•
Data reported in this paper will be shared by the lead contact upon request.
•
This paper does not report original code.
•
Any additional information required for data reported in this paper is available from the lead contact upon reasonable request.

[bib1] 1.Stramer B., Mayor R. Mechanisms and in vivo functions of contact inhibition of locomotion. Nat. Rev. Mol. Cell Biol. 2017;18:43–55. doi: 10.1038/nrm.2016.118. [DOI] [PubMed] [Google Scholar]

[bib2] 2.Lai E.C. Notch signaling: control of cell communications and cell fate. Development. 2004;131:965–973. doi: 10.1242/dev.01074. [DOI] [PubMed] [Google Scholar]

[bib3] 3.Banchereau J., Briere F., Caux C., Davoust J., Lebecque S., Liu Y.J., Pulendran B., Palucka K. Immunobiology of dendritic cells. Annu. Rev. Immunol. 2000;18:767–811. doi: 10.1146/annurev.immunol.18.1.767. [DOI] [PubMed] [Google Scholar]

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[bib6] 6.Kerppola T.K. Visualization of molecular interactions by fluorescence complementation. Nat. Rev. Mol. Cell Biol. 2006;7:449–456. doi: 10.1038/nrm1929. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[bib8] 8.Feinberg E.H., Vanhoven M.K., Bendesky A., Wang G., Fetter R.D., Shen K., Bargmann C.I. GFP Reconstitution Across Synaptic Partners (GRASP) defines cell contacts and synapses in living nervous systems. Neuron. 2008;57:353–363. doi: 10.1016/j.neuron.2007.11.030. [DOI] [PubMed] [Google Scholar]

[bib9] 9.Kim J., Zhao T., Petralia R.S., Yu Y., Peng H., Myers E., Magee J.C. mGRASP enables mapping mammalian synaptic connectivity with light microscopy. Nat. Methods. 2011;9:96–102. doi: 10.1038/nmeth.1784. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib10] 10.Tsetsenis T., Boucard A.A., Araç D., Brunger A.T., Südhof T.C. Direct visualization of trans-synaptic neurexin-neuroligin interactions during synapse formation. J. Neurosci. 2014;34:15083–15096. doi: 10.1523/JNEUROSCI.0348-14.2014. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[bib12] 12.Kinoshita N., Huang A.J.Y., McHugh T.J., Suzuki S.C., Masai I., Kim I.H., Soderling S.H., Miyawaki A., Shimogori T. Genetically encoded fluorescent indicator GRAPHIC delineates intercellular connections. iScience. 2019;15:28–38. doi: 10.1016/j.isci.2019.04.013. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib13] 13.Kinoshita N., Huang A.J.Y., McHugh T.J., Miyawaki A., Shimogori T. Diffusible GRAPHIC to visualize morphology of cells after specific cell–cell contact. Sci. Rep. 2020;10 doi: 10.1038/s41598-020-71474-0. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Fluorescent indicators for visualizing dynamic contact between cells and between processes originating from a single cell

Takashi Kanadome

Natsumi Hoshino

Susumu Jitsuki

Hidehiko Hashimoto

Takeshi Yagi

Takeharu Nagai

Summary

Graphical abstract

Highlights

Motivation

Introduction

Results

Design and characterization of the fluorescent indicator for visualizing cell-cell contact, termed Gachapin

Figure 1.

Visualization of dynamic cell-cell contact by Gachapin

Figure 2.

Application of Gachapin in neurons

Figure 3.

Visualization of contact between different cells and between processes originating from a single cell

Figure 4.

Application of Gachapin-C in neurons

Figure 5.

Discussion

Limitations of the study

Resource availability

Lead contact

Materials availability

Data and code availability

Acknowledgments

Author contributions

Declaration of interests

Declaration of generative AI and AI-assisted technologies in the writing process

STAR★Methods

Key resources table

Experimental model and study participant details

Cell culture

Animals

Method details

Plasmid construction

Plasmid transfection

Establishment of HEK293A stable cell lines

Cell imaging

Cell aggregation assay

Quantification and statistical analysis

Footnotes

Supplemental information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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