An assembly-regulated SNAP-tag fluorogenic probe for long-term super-resolution imaging of mitochondrial dynamics
Graphical abstract
Introduction
Mitochondria are generally considered to be the power source of cells and are key organelles for energy production, cell respiration, metabolic regulation, and signal transduction (Bereiter-Hahn and Vöth 1994; Westermann and Neupert 2000). It is known that mitochondrial dynamics are linked with cell fate. Mitochondria contact to form a network and their shape and positions change frequently and diversely in three dimensions, making real-time monitoring of mitochondrial dynamics in situ a challenging task (Gomes et al., 2011; Hoppins et al., 2007; McBride et al., 2006). For example, the fusion and fission of mitochondria are rapid and dynamic processes, which are generally considered to be closely related to apoptosis (Chan 2006). They interact with other organelles through membrane contact sites, vesicle transport, and signal transduction to regulate immune response, biosynthesis, energy metabolism, and cell turnover (Audano et al., 2018; Boldogh and Pon 2007; Wong et al., 2019).
Although electron microscopy (EM) with the nanometer-level resolution has been widely used to observe the different morphologies of mitochondria, it was not until the advent of fluorescence microscopy that dynamic observation of mitochondria in living cells became possible. Unfortunately, due to the existence of the diffraction limit, the spatial resolution of this far-field optical imaging method is limited to 200 nm (Hell 2007; Huang et al., 2010). In recent years, the emergence of super-resolution fluorescence microscopes such as stimulated emission deletion (STED) (Huang et al., 2013), structured illumination microscopy (SIM) (Huang et al., 2018; Schermelleh et al., 2008) and stochastic optical reconstruction microscopy (STORM) (Rust et al., 2006) has broken the resolution limit of conventional far-field microscopy, making it possible to observe the dynamics of mitochondria at the nanometer level. Among them, SIM implemented by illuminating with multiple interfering beams of light can achieve a spatial resolution of 100 nm, and has better biocompatibility and no additional requirements for the preparation of experimental materials. So far, it has become one of the key technologies in cell biology, especially in researching the dynamic changes of mitochondria. However, the realization of long-term super-resolution imaging of mitochondria dynamics requires accurate and stable labeling (Takakura et al., 2017), which is still extremely challenging. Compared with fluorescent proteins, organic small molecule fluorophores have higher brightness and better photostability, and have the potential to be used for mitochondria labeling. Zhang's group used a commercial small molecule organic fluorescent dye Atto 647N to stain mitochondria in living cells, and observed the interaction between mitochondria and lysosomes through dual-color SIM imaging (Han et al., 2017a). Diao and his colleagues proposed a strategy to discover and screen drugs by observing the interaction between mitochondria and lysosomes on the nanoscale, in which the mitochondrial staining dye they used was Mito-Tracker Green (Chen et al., 2019). Most mitochondrial probes are essentially lipophilic cationic dyes that depend on the electrostatic interactions with mitochondria. In this case, the accuracy and robustness of the labeling largely depend on the mitochondrial membrane potentials (Smith et al., 2011; Zielonka et al., 2017). Once the mitochondrial membrane potential changes, it will inevitably cause the dye to dissociate from the mitochondria, which in turn leads to an increase in the intracellular background. Therefore, the existing mitochondrial probes are still difficult to meet the requirements of long-term mitochondrial super-resolution imaging, especially those processes where the mitochondrial membrane potential changes.
SNAP-tag is a 20-kDa engineered mutant of the human repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) which rapidly and specifically reacts with para-substituted O6-benzylguanine (BG) derivatives by transferring the substituted benzyl group to its reactive site through a nucleophilic substitution reaction while releasing free guanine. It has been widely used as a protein self-labeling tag via genetical encoding (Brun et al., 2009; Gautier et al., 2009; Keppler et al., 2003; Leng et al., 2017; Srikun et al., 2010; Zhou et al., 2019). The O6-benzylguanine (BG) derivative probes can specifically react with SNAP-tag through the formation of a stable thioether bond to realize the labeling of the fusion protein. Therefore, labeling mitochondria with SNAP-tag fluorescent probes overcomes the shortcomings that changes in mitochondrial inner membrane potential will release mitochondrial probes based on electrostatic interactions. While a list of SNAP-tag probes have been developed, extensive washing processes are still required to reduce background fluorescence from unreacted or location-unspecific probes (Komatsu et al., 2011). These fussy washing processes are time-consuming and limit the application of probes in real-time imaging. So, fluorogenic probes, which exhibit significant fluorescent enhancement after labelling SNAP-tag, are urgently needed (Liu et al., 2014; Qiao et al., 2017; Sun et al., 2011).
Herein, we reported a novel naphthliamide-derived SNAP-tag fluorogenic probe AN-BG for long-term super-resolution imaging of mitochondrial dynamics (Scheme 1). Naphthalimide is the traditional environment-sensitive and two-photon fluorophore with large stokes shift and moderate brightness. The azetidinyl group was introduced to be in a flat conformation with the naphthalimide fluorophore, which can effectively suppress twisted intramolecular charge transfer and then effectively improve the brightness and photostability (Grimm et al., 2015; Liu et al., 2016). Meanwhile, this planarized molecular structure is conducive to the formation of J-aggregates which would quench the fluorescence (Scheme 1). After the probe is selectively covalently attached to SNAP-tag, the probe aggregates are separated and the fluorescence is then restored. Therefore, the aggregation and disaggregation of the probes control the changes of fluorescence, and the specific binding of the probe to SNAP activates the generation of fluorogenicity. Further, fluorescent labeling mitochondrial inner membrane proteins via SNAP tags overcomes the shortcomings that variations in mitochondrial inner membrane potential will release probes attached to mitochondria by electrostatic interactions. Therefore, AN-BG realized the stable labelling of mitochondria and the long-term imaging of mitochondrial dynamics under super-resolution microscopy.
Section snippets
Materials
Unless otherwise specifically stated, all reagents were purchased from commercial suppliers (Sigma-Aldrich, J and K, Innochem and Aladdin) and used without further purification. Solvents [dimethyl sulfoxide (DMSO), dimethylformamide (DMF), methanol] were purchased from J&K and used without further treatment or distillation. Silica gel (200–300 mesh) was purchased from Innochem. Unless otherwise specifically stated, Plasmids used in this paper were all purchased from Addgene.
Kinetic study of the reaction of AN-BG and SNAP-tag protein in vitro
To monitor the
Disassembly-driven turn-on fluorescence
The absorption and fluorescence spectra of AN-BG in different solvents were firstly investigated (Fig. 1 and Fig. S1). Since the presence of azetidine ring inhibited the TICT process, the quantum yields of AN-BG in all organic solvents were very high, in the range of 0.7–0.9 (Table 1). Due to the intra-molecular charge transfer process, as the polarity increased, the absorption wavelength and fluorescence wavelength gradually red-shifted. It was very significant that the fluorescent brightness
Conclusions
In summary, we reported a fluorescent probe for protein labeling and no-wash imaging to achieve super-resolution imaging in mitochondria and nuclei. Because this probe has good brightness, photostability and excellent biocompatibility, after being labeled, the long-term dynamic process of mitochondria can be tracked and observed in real time under SIM imaging, including mitochondrial fusion and fission, as well as the process of separation after rapid contact between mitochondria. Two-color SIM
CRediT authorship contribution statement
Wenjuan Liu: synthesized the probe and examined optical properties, did the cell imaging, wrote the paper. Qinglong Qiao: synthesized the probe and examined optical properties, wrote the paper. Jiazhu Zheng: synthesized the probe and examined optical properties. Jie Chen: did the cell imaging. Wei Zhou: did the cell imaging. Ning Xu: synthesized the probe and examined optical properties. Jin Li: synthesized the probe and examined optical properties. Lu Miao: did the cell imaging. Zhaochao Xu:
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We are grateful for the financial support from the National Natural Science Foundation of China (22078314, 21878286, 21908216), Dalian Institute of Chemical Physics (DICPI201938,DICP I202006), Dalian Talent Support Program (2018RQ16) and Liaoning Provincial Fund (2019JH2/10300016).
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