Introduction
Intercellular communication is essential for tissue homeostasis, specific cell functions, and responses to external stimulus. Cell–cell communications typically occur through various processes, such as diffusible factors, including cytokines and chemokines, secreted microvesicles, and direct passage through gap junctions [1]. Recently, a new impressive intercellular communication pathway was found that allows long-distance cell–cell contacts via tunneling nanotubes (TNTs) between these cells, which was initially reported in rat pheochromocytoma (PC12)-derived cells [2] and later in other types of cells [2–7]. The TNTs connecting two cells exhibit long tubular structures with diameters of 50–1500 nm and lengths of tens to hundreds of microns, which maintain a continuous cytoplasm membrane between the connecting cells, thus allowing the transportation of numerous cellular components, such as proteins, RNAs, viruses, and organelles, from one cell to another [8–10]. Generally, actin is included in the central composition and a membrane is wrapped outside, which is typically part of the cell membrane. Additionally, TNTs play distinct roles in the modulation of cell death involved not only in the delivery of injured cells [9–12] but also in enhancing the lysis of distant cells [13]. Thus, addressing the dynamic changes of such TNTs will be helpful for understanding the exact events of intercellular communication behaviors.
In recent years, studies on TNTs were performed in normal cells as well as on the pathogenesis of neurodegenerative diseases and cancer [14] mainly via electron and fluorescence microscopy [11,15–19]. However, it is difficult to observe ultra-fine TNTs in vivo because of their fragility to light, mechanical stress, or chemical fixation and the limit by the inherent shortcomings of the imaging techniques used. Although electron microscopy is able to provide spatial resolution as high as up to a nanometer, it cannot be used to image live cells [3]. In contrast, optical microscopies can be used to image live cells, but the spatial resolution is less than 200 nm owing to the optical diffraction limit. Since the first report on TNTs, only two kinds of markers targeting tube-like structures have emerged, including wheat germ agglutinin (WGA)-chromophore binding for cell membrane and phalloidin-chromophore targeting for F-actin. Both derivatives are expensive and difficult to purify [3,20,21]. Stochastic optical reconstruction microscopy (STORM) [22–24] is an advanced super-resolution imaging technique that offers promising potential to carry out biological imaging of dynamic TNTs between live cells. Together with special optical probes, it is possible to perform STORM imaging of dynamic TNTs between live cells. However, the lack of excellent STORM probes makes it difficult to observe the ultra-fine structures of TNTs in the natural environment. Until now, there have been no reports on super-resolution imaging of dynamic TNTs between live cells.
In this study, we designed a simple cyanine-based probe to label TNTs and to perform STORM imaging. The probe was composed of a Cy5 dye with a two-ester moiety at the N-position (Fig. 1). It was characterized using nuclear magnetic resonance (NMR) and mass spectra (See Supplementary Information). Then, the probe was used to label the TNTs and carry out STORM imaging to study the cleavage of the TNTs and the ultra-fine details of dynamic changes.
Experimental
First, we synthesized probe 1 via a simple reaction method with high yields. In detail, 1 was prepared by heating 2,3,3-trimethylindolenine and an equivalent amount of 4-bromobutyric acid ethyl ester in acetonitrile solution. The obtained quaternary ammonium salt was subsequently heated with condensation reagent in Ac2O/NaOAc for 30 min. The obtained blue residue was further purified using a silica column, and the synthetic details and structure identification of the probe and intermediate are provided in the Supplementary Information [25–27].
Discussion
The photophysical behaviors of 1 were tested to confirm the feasibility of the probe for TNT imaging. The absorption and fluorescent emission spectra of 1 were measured in ethanol to show the peaks at labs = 654 nm and lem = 679 nm, respectively (Figs. 2(a) and S1), indicating that the probe can be excited at 656 nm wavelength.
Fig.2 Photophysical properties of probe 1 were evaluated. (a) Excitation and emission spectra of probe 1 (black and red curves, respectively), with a laser excitation wavelength of 633 nm. (b) Duty cycle calculated for probe 1. (c) Single-molecule fluorescence time traces measured in the absence of bME and oxygen-scavenging system |
A home-built STORM optical setup was used to measure the transient photoblinking of 1 under illumination of a single laser beam (656 nm) with a power density of 0.682 kW/cm2. The marked photoblinking of 1 was observed with a high photon number (6000–8000) and larger on–off duty cycle (ca. 0.00126) than that of Alexa 647 [26]. It could be switched between fluorescence on and off states and remained in the dark state for a sufficiently long time, which is suitable for single molecule localization-based STORM imaging. Additionally, the duration of photoblinking of probe 1 could last for more than 800 s with a suitable switching frequency (Fig. 2(c)), and over 60% of probe molecules survived after a 700 s illumination period (Fig. 2(b)), which made it possible for long-term observation of the dynamic behaviors of live cells.
The cytotoxicity of probe 1 was further studied to assess the impact on the dynamics of TNTs, including the formation or cleavage processes. HeLa cells were cultured with 1 at different concentrations (1.0, 2.0, and 4.0 mmol/L) for different time periods (1, 5, and 10 h) (Fig. 3(e)). Most of the cells survived and were active at the concentration of 4.0 mmol/L for even 10 h of incubation. This showed that the probe exhibited rare toxicity to HeLa cells, which was favorable for live cell imaging. As biological dynamic changes in live cells usually last for several minutes, a fluorescent probe without cytotoxicity would be extremely important to the study of natural biological processes.
Fig.3 Fluorescent imaging of the probe in HeLa cells. The co-localization experiment was conducted by co-incubating 1 (1 mmol/L) (a) with a commercially available dye, membrane tracker-Dioctadecylcarbocyanines (DIO) (1 mmol/L) (b). The merged image (c) and Pr coefficient (d) was obtained by overlapping (a) and (b); FITC is the abbreviation of fluoresceinthioisocyanate. (e) Cell viability experiment on HeLa cells when incubating with probe 1 |
After accomplishing all primary tests, super-resolution imaging of TNTs was further performed on the STORM setup using HeLa and COS-7 cells to identify the exact location of 1 in the live cells. After the probe was incubated with COS-7 cells for 15 min, the sample was washed with phosphate buffer saline (PBS) and then placed on the sample stage of the STORM system to check the photoblinking in live cells. The specimen was illuminated with a single laser beam at 656 nm with a power density of 200 mW/cm2. As shown in Figs. 4(a) and S2(a), according to the wide-field image, the fluorescent probe mainly stained the long tube between two COS-7 cells but could not provide fine details of the TNTs. The photoblinking images of the probe were recorded using an electron multiplying charge coupled device (EMCCD, 60 Hz). A super-resolved image was reconstructed using 200 frames with a reconstruction algorithm of the fast localization algorithm based on the continuous-space formulation (FALCON) for high-density super resolution [28]. As shown in Figs. 4 and S2, we acquired the STORM images of the TNTs between COS-7 cells, exhibiting thin and long tube structures. Compared with the wide-field images, the STORM images showed a clear linear structure of the TNTs, which could be used to quantify the thickness of the tubes.
To further disclose the TNT dynamic change, the FALCON algorithm was used to reconstruct the dynamic STORM images of the TNTs using 180 frames with a step gap of 20 frames (3 s temporal resolution). As demonstrated in Figs. 4(a) and S2(b)–S2(f), a consecutive change in the TNT with ultra-fine structures was obtained to show the detailed changes of TNT cleavage. From the magnified image of the region of interest, as indicated by the dashed box in Figs. 4(b) and 4(c), a clear cleavage of the TNTs and some interesting features could be observed (see Electronic Supplementary Videos S1 and S2). First, the breakage of the TNTs started at 46 s from the right end; and on the left side of the TNT, it slowly shrunk and finally formed a circle (Fig. 4(b)). A zoomed in picture of the cleavage site of TNT is shown in Fig. 4(c), from which we observed the initial TNT to be a linear structure, and later, the breaking site showed up with a circle formed at the right end with an increase in diameter of the circle. Meanwhile, cleavage occurred at the left end of the circle. In the wide-field image, the circle ring is totally a solid flat disc with a diameter of 530.1±14.1 nm, which is considerably larger than the optical diffraction limit; however, in the STORM image, the solid circle became a hollow ring with an full width at half maximum (FWHM) value of 133.5±1.5 nm, as calculated by Gaussian fitting (Figs. 4(d) and 4(e)). This provides a promising chance to study the biological functions of TNTs.
Fig.4 STORM imaging of TNTs and their dynamic changes. (a) One snapshot (t = 0 s) from a 184-s movie (left) and a reconstructed STORM image from 180 frames recorded over 3 s. (b) and (c) Time-series STORM snapshots of intercellular filament dynamics. Each image was reconstructed from 180 frames recorded over 3 s. (d) Magnified images of the circle in the square in (b) obtained by wide-field and STORM imaging. (e) Fluorescence intensity profiles along the white lines in (d). The black line shows the signal in the STORM image, whereas the blue line shows the signal in the wide-field image. FWHM values of these profiles are 133.5±1.5 nm (super-resolution image) and 530.1±14.1 nm (wide-field image), respectively |
Conclusions
We developed a simple Cy5-based probe for STORM imaging of intercellular tunneling nanotubes. The probe, which has two ethyl-butyrate groups that can be used as TNT membrane binding sites, kept photoblinking and remained photostable for long-term STORM imaging. Using this probe, the typical TNT ring-like structure with a diameter of 133.5±1.5 nm formed during the cleavage of the TNTs was observed by STORM imaging, which provided the first view of TNTs cleaving in live cells. To the best of our knowledge, this is the first report on a simple probe for STORM imaging of TNT dynamics, particularly for the analysis of its formation and functional roles in cell communication.