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. 2021 Jul 28;7(8):1400–1407. doi: 10.1021/acscentsci.1c00645

Reconstructing Soma–Soma Synapse-like Vesicular Exocytosis with DNA Origami

Jiangbo Liu †, Min Li †,*, Fan Li †, Zhilei Ge ‡, Qian Li ‡, Mengmeng Liu §, Jiye Shi ∥, Lihua Wang ∥,⊥, Xiaolei Zuo †,‡,*, Chunhai Fan ‡, Xiuhai Mao †,*
PMCID: PMC8393203  PMID: 34471683

Abstract

graphic file with name oc1c00645_0005.jpg

Cell–cell communications exhibit distinct physiological functions in immune responses and neurotransmitter signaling. Nevertheless, the ability to reconstruct a soma–soma synapse-like junction for probing intercellular communications remains difficult. In this work, we develop a DNA origami nanostructure-based method for establishing cell conjugation, which consequently facilitates the reconstruction of a soma–soma synapse-like junction. We demonstrate that intercellular communications including small molecule and membrane vesicle exchange between cells are maintained in the artificially designed synapse-like junction. By inserting the carbon fiber nanometric electrodes into the soma–soma synapse-like junction, we accomplish the real-time monitoring of individual vesicular exocytotic events and obtain the information on vesicular exocytosis kinetics via analyzing the parameters of current spikes. This strategy provides a versatile platform to study synaptic communications.

Short abstract

A soma−soma synapse-like junction was constructed via DNA origami nanostructures. A carbon fiber nanoelectrode was inserted into the junction for vesicular exocytotic monitoring.

Introduction

Intercellular communications play crucial roles in living organisms for normal physiological functions.1 For instance, in the immune system, to induce the cytolysis of cancer cells, the soma of a CD8+ T cell will release perforin, granzymes, and granulysin to the cell membrane of the cancer cell after the T cell accomplishes the recognition and binding for the cancer cell.2,3 Besides, soma–soma synaptic junctions are routinely described based on previous studies;4−6 for example, neuronal soma can release adenosine triphosphate to activate the P2X7 receptors on the tightly enwrapped satellite glial cells, accomplishing the intercellular communications between neurons and satellite glial cells via a soma–soma interaction.4 Hence, the artificial reconstitution of a soma–soma synapse-like junction with the normal physiological interaction between cells provides a model system to better understand intercellular communications. However, the reconstruction of a controllable soma–soma synapse-like junction in vitro to mimic the multicellular system in vivo remains a technical challenge.7−11

To date, methods devoted to construct a synapse have mainly relied on the inherent growth characteristics of cells to form intercellular junctions. For example, a Petri dish culture or three-dimensional (3D) culture has been used to form glial–neuronal cocultures models.12 However, the elongated and branched structure of neurons greatly affects the directionality and controllability of the artificial synapse.13 As an alternative, researchers have used microfluidic channels to spatially separate the soma and elongated axons to control the direction of axon elongation.14,15 The spontaneous oriented elongation of neurons along the microfluidic channel could form ordered neuromuscular junctions, greatly improving the regularity of intercellular junctions. However, replication of nature-evolved processes to engineer the controllable soma–soma synapse-like junction in vitro has remained elusive until now.

With its high controllability and precision,16,17 the DNA nanostructure has been extensively exploited to engineer internanoparticle18,19 or intercell interactions20−22 in a programmable manner with high efficiency. Herein, we reconstruct a soma–soma synapse-like junction by using DNA origami nanostructures (DONs) to conjugate rat phaeochromocytoma (PC12) cells, which are a type of glial cell with neuronal vesicular exocytotic activity.23 The soma–soma synapse-like junction was visually characterized by atomic force microscopy (AFM) and scanning electron microscopy (SEM). We achieved the real-time amperometric monitoring of vesicle exocytosis by inserting carbon fiber nanoelectrodes (CFNEs) into the soma–soma synapse-like junction. We anticipate that the DON-mediated soma–soma synapse-like junction will provide a new perspective on constructing synapses for the research of synaptic communications.

Results and Discussion

Reconstruction of a Soma–Soma Synapse-like Junction

Six-helix bundle (6HB) DNA origami nanostructures24 that contain asymmetrically appended single-stranded DNA (ssDNA) overhangs were chosen as linkers to conjugate PC12 cells (Figure 1a). The 6HB DON was assembled by using hundreds of short staples to fold a long ssDNA scaffold into an approximately 400 nm monomer nanostructure with linear tomography (Figures S1 and S2). Considering that the cells are on the micron scale, we prolonged the length of 6HB DONs to the micrometer scale by designing outstretched strands on the end of a 6HB monomer. With the specific Watson–Crick base pairing, the micrometer end-to-end linked 6HB DONs could be formed. As visually characterized by AFM, we found that the prolonged 6HB DONs showed more curved configurations than the 6HB monomer (Figure 1b, Figure S3), indicating that the prolonged 6HB DONs possessed a higher flexibility than the monomeric 6HB DONs (for sequences and the method, see the Experimental Section in the SI and Tables S1 and S2). In detail, we found that the ssDNAs on the two sides of Janus DONs could be visualized in an AFM image (Figure 1b). This result indicated that ssDNAs were asymmetrically stretched out from Janus DONs, facilitating the further cell conjugation.

Figure 1.

Figure 1

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Cell conjugation and soma–soma synapse-like junction construction with DONs. (a) Scheme for cell conjugation with DONs. After incubating the mixed cells (anchoring of the chol-DNA on cell membranes) with the DONs, the cell conjugation could be formed. (b) Design and AFM image of Janus DONs. Scaffold M13 and link strands are shown in blue, while green and red staple strands on the two side of Janus DONs were used to hybridize with chol-DNA on cell membranes. The white arrow in the AFM image is for appending staple ssDNA strand overhangs. Scale bars: 200 nm in the zoomed out picture and 20 nm in the zoomed in picture. (c) CLSM image of a PC12 cell membrane after anchoring 10 μM chol-DNA strand hybridized with fluorescently labeled complementary strands. Scale bar, 10 μm. (d) CLSM image of a PC12 cell membrane after anchoring 1 nM Janus DONs hybridized with fluorescently labeled complementary strands. Scale bar, 10 μm. (e) Flow cytometry and CLSM image of cell conjugation with DONs. Scale bar, 50 μm. (f) CLSM image of a soma–soma synapse-like junction. Scale bars: 50 μm in the zoomed out picture and 10 μm in the zoomed in picture.

To conjugate cells with DONs, PC12 cell membranes were first functionalized with cholesterol-modified DNA (chol-DNA; for sequences and the method, see the SI, Table S3) strands which were complementary to the overhangs on Janus DONs. To verify the anchoring of chol-DNA on the cell membrane, single-stranded complementary DNA modified with Alexa 647 was used to hybridize with the chol-DNA. As imaged by confocal laser scanning microscopy (CLSM, Figure 1c), strong Alexa 647 signals were observed on cell membranes, indicating that chol-DNA could be effectively anchored on the cell membrane with high density. Flow cytometry also confirmed the effective membrane anchoring of chol-DNA (Figure S4). To evaluate the recognition capability of Janus DONs anchored on the cell membrane, we first labeled the Janus DONs by half hybridizing the overhangs with fluorescently modified complementary strands. Then we incubated the fluorescence labeled Janus DNA with cells modified with chol-DNA on the cell membrane. As shown in Figure 1d, asymmetrical fluorescence patches on the cell membrane were obtained in CLSM image, demonstrating that DONs were nonuniform distributed on cell membranes. To analyze the asymmetrical patchy distribution mechanism of DONs on the cell membrane, we next compared the curvatures of DONs and great circle of spherical cells. We found that the curvatures of DONs were relatively larger than that of the great circle of spherical cells (Figure S5). These results indicated that DONs were not concentrated on the “equatorial-radius” of the spherical cells. Instead, DONs probably located on specific locations on the cell membrane, the curvatures of which can match well with that of DONs (Figure S6). As a consequence, DONs were distributed on the cell membrane in an uneven way. And these fluorescence patches of DONs on the cell membrane indicated that DONs could hybridize with the DNAs anchored on the cell membrane, facilitating the further cell connection.

To investigate the anchoring stability of DONs on the cell membrane, we compared DONs conjugation system to simple single-stranded chol-DNA conjugation system, where ssDNAs were used to conjugate two cells by hybridization. Notably, we found that single-stranded chol-DNA anchored on the cell membrane tended to be internalized after cells were cultured at 37 °C for 2 h (Figure S7), indicating that DONs showed a higher membrane anchoring stability. To evaluate the effect of chol-DNA and DONs on cell activity, we then performed real-time cellular analysis (RTCA, Figure S8). We found that negligible alterations of cell growth curves were obtained, demonstrating that the anchored chol-DNAs and DONs showed a negligible effect on cell proliferation.

To accomplish the connection of PC12 cells, two groups of equivalent cells were first separately stained with different fluoresceins (CellTraker Green and CellTraker Deep Red, separately). Then, the two fluorescently stained cells were modified with different chol-DNAs which could hybridize with the DONs’ asymmetrical overhangs. Incubating the mixed cells with the DONs bearing matched overhangs, a two-cell conjugation pattern could be formed. Flow cytometry analysis was used to evaluate the cell conjugation capability of DONs. Notably, we found that approximately 43% of cells were conjugated by DONs (Figure 1e). As a sharp contrast, a negligible two-cell connection was obtained without DONs (∼2%, Figures S9 and S10). In addition, we compared the efficiency of the DON cell conjugation to that of the simple single-stranded chol-DNA conjugation system. Similarly, we obtained negligible two-cell connections in the simple single-stranded chol-DNA conjugation system (∼3%, Figure S11). The above results indicated that DONs possessed a higher cell conjugation capability than the simple single-stranded chol-DNA conjugation system. Then, we visually characterized the two-cell conjugation patterns before and after cell adhesion by CLSM (Figure 1e,f). Prominently, we found that the DON-based conjugating strategy could greatly improve the possibility of cell adherence in the conjugation state, facilitating the formation of a soma–soma synapse-like junction (Figure 1f).

Intercellular Communication between a Soma–Soma Synapse-like Junction

We next investigated intercellular communication between a soma–soma synapse-like junction. Based on a previous study, a gap junction could be established upon the PC12 cell conjugation.22 To verify the gap junction-mediated cell–cell communication of small molecules, we exploited CLSM to visualize the communication of small molecules in conjugated cells (Figure 2a). First, the conjugated cells were prestained with CellTraker Deep Red dye and CellTraker Green dye which are types of small-molecule dyes. After the conjugated cells were cultured for 12 h, we observed yellow fluorescence plaques in the conjugated cells (Figure 2a), indicating the normal intercellular communication of small molecules between the soma–soma synapse-like junction. Further, to verify the intercellular communication of membrane vesicles probably mediated by a tunneling-nanotube,22,25−27 we stained the two preconjugated PC12 cell membrane vesicles with two different-colored lipid fluorescent dyes, DiO (green) and DiI (red), separately (Figure S12). Next, we investigated the intercellular trafficking of membrane vesicles by using CLSM imaging. After 8 h of cell incubation, we observed the speckles of mixed color (yellow) in the two conjugated cells (Figure 2b, Figure S13). With an extension of the incubation time, the membrane vesicle transfer increased (Figure 2c, more details in the Experimental Section in the SI). Notably, we found that approximately 40% of the membrane vesicles were transferred to the conjugated cell after 8 h of coculture (Figure 2c), indicating the normal intercellular exchange of membrane vesicles between conjugated cells. Altogether, these results demonstrated that intercellular communication between the soma–soma synapse-like junction was implemented.

Figure 2.

Figure 2

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Cell–cell communication between soma–soma synapse-like junction. (a) CLSM image of small molecules transporting between soma–soma synapse-like junction. Scale bars: 20 μm in zoomed out picture and 10 μm in zoomed in picture. (b) CLSM image of DiO and DiI stained membrane vesicle transporting between soma–soma synapse-like junction. Scale bar, 20 μm. (c) Quantification of DiO membrane vesicles transfer. Data are presented as the mean ± s.e.m. from three independent experiments.

Characterization of CFNEs

To further investigate the intercellular communication between a soma–soma synapse-like junction, we attempt to monitor the vesicle exocytosis by inserting a CFNE into the nanogap between two PC12 cells (Figure 3a). To this end, we first constructed CFNEs with defined nanotips by using flame-etched carbon fibers coupled with glass-pulled nanopipette microfabrication (Figure S14a).28−30 To characterize the electrochemical property of CFNEs, we scanned cyclic voltammetric curves in K3[Fe(CN)6] solution. Prominently, we observed the well-defined sigmoid-shaped voltammograms, demonstrating the nonlinear diffusion characteristic of CFNEs. It is worth noting that approximate equivalent limiting current plateaus were obtained by different CFNEs (Figure S14b), indicating the excellent repeatability and controllability of CFNE fabrication. Further, the CFNEs were visualized by SEM imaging. We found that the lengths of carbon fibers were approximately 25 μm (Figure S15), and the nanotips were ∼100 nm in diameter (Figure 3b).

Figure 3.

Figure 3

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CFNEs inserting inside a soma–soma synapse-like junction. (a) Schematic illustration of CFNEs inserting into a soma–soma synapse-like junction. (b) SEM image of a CFNE nanotip. Scale bar, 100 nm. (c) AFM image of a soma–soma synapse-like junction. Scale bars: 10 μm in the left picture and 2 μm in the right picture. (d) SEM image of a soma–soma synapse-like junction. Scale bars: 2 μm in the left picture and 400 nm in the right picture. (e) Statistical analysis for sizes of CFNE nanotips and junction clefts in the SEM image. (f) Bright-field photomicrograph showing CFNE inside a synapse-like junction. Scale bar: 5 μm. (g) Limiting reduction current plateaus of K3[Fe(CN)6] for CFNEs at different states of its nanotip inside a soma–soma synapse-like junction.

To further demonstrate that the sizes of CFNEs are suitable enough for soma–soma synapse-like vesicular exocytosis monitoring, we characterized the nanogaps of a soma–soma synapse-like junction with AFM and SEM (Figure 3c,d). We found that the junction cleft was approximately 150 nm, which was larger than the nanotips of CFNEs (∼100 nm) (Figure 3e), illustrating that inserting nanotips into the junction cleft is feasible. We then inserted CFNE into the soma–soma synapse-like junction via a micromanipulator coupled with a microscope (more details in the Experimental Section in the SI), and typical images are shown in Figure 3f and Figure S16.

To further verify that CFNEs were not destroyed during the insertion process, we performed a series of controlling the state of short CFNEs (cyclic voltammetry characterization in Figure S17) at different insertion depths inside the junctions in K3[Fe(CN)6] solution (Figure 3g). By amperometrically recording the limiting K3[Fe(CN)6] reduction current plateaus of CFNE via a patch clamp, we found a ∼40% current decrease when half of the CFNE was inserted into the soma–soma synapse-like junction. Notably, a negligible current was obtained when CFNE was fully inserted into the soma–soma synapse-like junction, indicating the satisfactory sealing of the junction around the CFNE. After withdrawing the whole electrode from the synapse-like junction, the currents plateau was approximately recovered to its original value. Altogether, these results demonstrated that the insertion process did not affect the electrochemically active surface of CFNEs. Considering that the junction is extremely small (∼150 nm), the insertion process probably leads to being somewhat invasive/disruptive to the junction.

Monitoring Vesicular Exocytosis within a Soma–Soma Synapse-like Junction

After CFNEs were inserted into the soma–soma synapse-like junction, vesicular exocytosis and neurotransmitter release were monitored via amperometry (Figure 4a). Based on previous studies, the majority of the neurotransmitter contained in PC12 cells is dopamine (divalent oxidation–reduction reaction).23,31 After stimulating the cell with high-K+ solution, we recorded quantal amperometric spikes via a patch clamp under the potential of 700 mV. This result indicated that instantaneous vesicular exocytosis and dopamine releasing events occurred within the soma–soma synapse-like junction (Figure S18).

Figure 4.

Figure 4

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Analysis of vesicular exocytosis within a soma–soma synapse-like junction. (a) Scheme for the two modes of vesicular exocytosis and dopamine releasing: K&R and FCF. (b) Schematic illustration of a typical amperometric spike for analyzing the number of dopamine and vesicular exocytotic dynamics. (c, i) Histogram showing the distribution of Nmolecules. (c, ii) Scatter plots showing durations (t1/2) and number of released dopamine molecules (Nmolecules) for each simple event (blue) and complex event (red). (c, iii) Histogram showing the distribution of durations (t1/2). (d) Average Nmolecules and (e) average t1/2 (data obtained from the analysis shown in part c). (f) Rise time and fall time from peak analysis. Data are presented as the mean ± s.e.m. *P < 0.05, **P < 0.01.

To analyze in detail the characteristics of the releasing events in a soma–soma synapse-like junction, we defined two typical amperometric peaks, a “simple event” and “complex event”, respectively (Figures S19 and S20).30 The simple event was a single rising spike with the first derivative of the current trace spike (dI/dt) 5-fold larger than the root-mean-square (r.m.s.) noise, and the complex event contained at least two subspikes with the dI/dt 3-fold larger than the r.m.s. noise. Both events were separated from the previous and following events by at least 40 ms (more details in the Experimental Section in the SI). Statistically, we found that approximate 82.6% (247 out of 299) of spikes were simple events, with the remaining ∼17.4% (52 of 299) of events being complex events. These results indicated that the neurotransmitter exocytosis inside the DON-mediated soma–soma synapse-like junction mainly relied on the simple release event. Based on previous studies,32−34 simple events were attributed to the results of full-collapse fusion (FCF), releasing the full vesicular contents outside the cell one time with the vesicle membrane completely fused into the plasma membrane. The complex events may be the result of “Kiss and Run” (K&R), releasing vesicular contents several times with transient membrane fusion and retrieval. Thus, we demonstrated that the FCF-based release was preferred in the soma–soma synapse-like junction as compared to the K&R mode. Furthermore, as shown in Figure 4b, we analyzed the number of the released dopamine molecules (Nmolecules) by calculating the integral area of the amperometric spikes. We found that the numbers of released molecules were 2.17 ± 0.12 × 105 and 3.53 ± 0.54 × 105 for simple events and complex events, respectively (Figure 4c,d), which were 2-fold more compared to previous studies.35 The increased numbers of released dopamine between the soma–soma synapse-like junction indicated that vesicles released in the synapse-like junction may be more active than vesicles in the single cell, which facilitated the intercellular communication.

We further evaluated vesicular exocytotic dynamics within the soma–soma synapse-like junction by analyzing the t1/2 of amperometric spikes. We found that t1/2 values were 0.31 ± 0.01 and 0.98 ± 0.09 ms for simple events and complex events, respectively (Figure 4c,e). The approximately 2-fold longer time indicated the longer release durations of the complex event. Further, as shown in Figure 4b, the time from 25% to 75% of the maximum magnitude on the ascending part, which illustrated the opening time of the vesicle, was defined as the rise time trise, and the time from 75% to 25% on the descending part, which illustrated the closing time of the vesicle, was defined as the fall time, tfall. Statistically, we found that shorter trise and tfall values (0.15 ± 0.02 and 0.16 ± 0.04 ms, respectively, Figure 4f) were obtained compared to previous works,35 indicating that the vesicle opening and closing times inside the soma–soma synapse-like junction were faster than those on a single cell soma. This is probably attributed to the fact that vesicles released in the synapse-like junction are required to be more active for accomplishing the normal intercellular communication.

Conclusions

In summary, we developed a DON-based strategy for PC12 cell conjugation and soma–soma synapse-like junction reconstitution. We found that normal intercellular interactions were maintained after cell conjugation, such as the exchange of small molecules and membrane vesicles. By inserting CFNE into the nanogap within the soma–soma synapse-like junction, we achieved the monitoring of chemical synaptic transmission between two conjugated PC12 cells. Additionally, we found that vesicles released between the synapse-like junction were more active than those in a single cell. We wish that this novel strategy will be used for probing and quantifying vesicular exocytotic neurotransmitters releasing in synapses, offering a deep understanding of neuronal communications.

Acknowledgments

This study was financially supported by the National Key R&D Program of China (Grants 2018YFA0902600), NSFC (Grants 21804091, 21904086, and 21804088), “Shuguang Program” supported by the Shanghai Education Development Foundation and Shanghai Municipal Education Commission (Grant 18SG16), Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (Grant 20171913), and Innovative Research Team of High Level Local Universities in Shanghai (SSMUZLCX20180701).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acscentsci.1c00645.

  • Additional data and figures including nanostructure models, AFM images, oligomer length values, flow cytometry results, curvature values, CLSM images, RTCA results, CV curves, SEM image, amperometric current traces, and DNA sequences (PDF)

The authors declare no competing financial interest.

Supplementary Material

oc1c00645_si_001.pdf (1.5MB, pdf)

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