Journal Pre-proof A highly speciﬁc Golgi-targetable ﬂuorescent probe for tracking cysteine generation during the Golgi stress response Xue Zhang, Caiyun Liu, Xinyu Cai, Bin Tian, Hanchuang Zhu, Yanan Chen, Wenlong Sheng, Pan Jia, Zilu Li, Yamin Yu, Shengyun Huang, Baocun Zhu
To appear in:
Sensors and Actuators: B. Chemical
2 November 2019
16 January 2020
3 February 2020
Please cite this article as: Zhang X, Liu C, Cai X, Tian B, Zhu H, Chen Y, Sheng W, Jia P, Li Z, Yu Y, Huang S, Zhu B, A highly speciﬁc Golgi-targetable ﬂuorescent probe for tracking cysteine generation during the Golgi stress response, Sensors and Actuators: B. Chemical (2020), doi: https://doi.org/10.1016/j.snb.2020.127820
A highly specific Golgi-targetable fluorescent probe for tracking cysteine generation during the Golgi stress response Xue Zhang,a Caiyun Liu,a,* Xinyu Cai,a Bin Tian,b Hanchuang Zhu,a Yanan Chen,a Wenlong Sheng,b,* Pan Jia,a Zilu Li,a Yamin Yu,a Shengyun Huang,c,* and Baocun Zhu a,* a
School of Water Conservancy and Environment, University of Jinan, Jinan 250022,
Biology Institute, Qilu University of Technology (Shandong Academy of Sciences),
Jinan 250103, China.
Department of oral and maxillofacial surgery, Shandong Provincial Hospital
Affiliated to Shandong University, Jinan 250021, China.
The probe could quantitatively and sensitively detect Cys (DL=5.1×10−8 mol/L)
The probe displayed prominent selectivity to Cys than other species including biothiols The probe could specifically target the Golgi apparatus
The probe could be applied to tracking basal Cys in live cells and zebrafish
The probe could track the generation of Cys during the Golgi stress response
in live cells and zebrafish
In the appropriate Golgi stress response, adaptive repair will occur by changing
or triggering the expression of related pathways. Furthermore, it also can inhibit the
cytotoxicity of cells in the altered redox homeostasis. Elevated biosynthesis and transport of cysteine (Cys) will be induced to restore the abnormal redox state, and
more evidences are needed to confirm this process in the Golgi stress response. Herein, we developed a novel Golgi-targetable Cys-specific fluorescent probe
GT-Cys for sensitively detecting Cys generation during the Golgi stress response. In probe GT-Cys, thiobenzoate moiety was chosen as recognition group of Cys, and 4-CF3-7-aminoquinoline dye was selected as fluorophore and Golgi targetable unit. Probe GT-Cys can selectively and sensitively respond to Cys. Additionally, it has good targetable properties, facilitating the study of complicated stress response of 2
Golgi apparatus. Importantly, the applications of probe GT-Cys in biological imaging showed that it is sensitive enough to basal Cys, especially to endogenous Cys during the Golgi stress response. Keywords: Fluorescent probe; cysteine; Golgi stress response; Golgi-targetable bioimaging; aminoquinoline; thiobenzoate
Organelles stress response is an essential part to meet the needs of cells,
including stress response of endoplasmic reticulum (ER), mitochondria, lysosome and
Golgi apparatus . In recent years, the mechanism of organelle stress response has
been found in detail, especially ER stress response. In contrast, Golgi stress response has not been studied extensively [2-6]. As a major organelle in most eukaryotic cells,
Golgi apparatus is a dynamic structure of proteins packing, transport and sorting .
When the cells are stimulated, they will be associated with the elevated Golgi stress response. At the same time, they will trigger the corresponding pathways to reduce
the burden of Golgi apparatus. However, excessive Golgi stress response may contribute to apoptosis . Therefore, the detailed mechanism of Golgi stress
response should be fully clarified. Recently, the mechanism of Golgi stress response induced by appropriate cell
stimulation to restore redox homeostasis has been further disclosed. It has been confirmed that cysteine (Cys) tends to increase during the Golgi stress response to provide cytoprotection . As the precursor of glutathione (GSH), Cys can participate 3
in the antioxidant defense system together with GSH to provide protection for cells . The redox state in cells is determined by the amount, production rate and consumption rate of reactive oxygen species (ROS) and reducing substances, as well as the interaction between these species . When oxidative stress occurs in living cells, redox equivalence can be restored by changing the production of Cys.
important to expound its cytoprotection functions.
Obviously, tracking the production of Cys during the Golgi stress response is very
As a non-invasive and high spatial resolution method, fluorescent probe has been widely used in the tracking of bioactive molecules in living cells and in vivo, and
provide an effective way to explore physiological and pathological functions of these
species [11-12]. In recent years, a large number of fluorescent probes have been developed to detect Cys by using its nucleophilic and reductive properties. The related
mechanisms include Michael addition, cracking reaction, cyclization reaction, and so
on [13-22]. Especially, organelle-targeted fluorescent probes are of great significance in detecting bioactive molecules in situ and dynamically. For instance, Fan’s group
developed a ratiometric and mitochondrial-targeted fluorescent probe to visualize Cys/Hcy . Ye’s group developed an ER-targeted fluorescent probe to study GSH,
Cys and HOCl in ER stress . Yin’s group developed a lysosome-targeted fluorescent probe to detect lysosomal cysteine in situ, in order to explore the relationship between apoptosis and lysosomal Cys . Although many outstanding probes have been used in Cys detection, there are still some aspects to be improved. First of all, it is a severe challenge to distinguish Cys from other biothiols such as Hcy, 4
GSH and H2S by fluorescent probes. Among them, some probes distinguish Cys from Hcy according to their different reaction rates with Cys and Hcy respectively. However, in the long-term reaction between the probe and Cys, the interference caused by Hcy cannot be avoided. Secondly, despite the wide applications of fluorescent probes in specific organelles [26-32], specific Golgi-targeting probes are rarely reported [33-46]. Especially, Cys, as one of the major biothiol in biological
system, its function in the Golgi apparatus deserves further study.
In this respect, we are committed to develop a novel fluorescent probe, which has the ability to target the Golgi apparatus, and can sensitively and selectively detect
endogenous Cys. In order to monitor Cys specifically, probe GT-Cys was constructed
by combining thiobenzoate with aminoquinoline derivative. The thiobenzoate moiety was selected as the recognition group because of its notable selectivity to Cys .
Aminoquinoline derivative was chosen as fluorophore because of its excellent
Golgi-targetability and convenient synthesis [41,45,46]. As expected, probe GT-Cys was successfully applied to the specific detection of Cys and has the satisfactory
feature of targeting the Golgi apparatus. In addition, based on the lower cytotoxicity, probe GT-Cys also has been utilized to monitor basal Cys in living cells and zebrafish.
Most importantly, probe GT-Cys can be served as a powerful tool to monitor the production of Cys under elevated Golgi stress.
2. Experimental section 2.1 General Unless otherwise stated, for fluorescence spectra measurements, the probe 5
GT-Cys was dissolved in ethanol for stock solution, and the test samples were obtained in the solution of PBS/ethanol (8:2, pH = 7.4, 10 mM PBS). Upon the addition of analytes, the fluorescence intensity of probe GT-Cys at 518 nm was recorded after 30 min at 25 °C, and the slit widths were 4 nm. Bioimaging experiments were carried out under confocal fluorescence microscope, selecting different type of cells and five-day-old zebrafish as samples. Furthermore, the
utilization of materials and instruments were detailed in the Supporting Information. 2.2 Synthesis of probe GT-Cys
3. Results and discussion 3.1 Optical properties of probe GT-Cys toward Cys Thiobenzoate, as a novel Cys receptor reported by Tang et al, was introduced to 6
specifically and sensitively recognize Cys via the nucleophilic addition reaction . On the other hand, 4-CF3-7-aminoquinoline as a Golgi-targeting group has been fully confirmed by several newly published studies [41,45,46]. With the benefits of enhanced intramolecular charge transfer (ICT) structure and relatively high fluorescence quantum yield, 4-CF3-7-aminoquinoline is an ideal candidate for constructing novel Golgi-targeting fluorescent probes. The possible detection
mechanism was also further confirmed by HRMS (see the Supporting Information)
and shown in Scheme 2. The fluorescence behaviors of GT-Cys (5 μM) to Cys were
tested in the solution of PBS/ethanol (8:2, pH = 7.4, 10 mM PBS). The probe solution
exhibited very weak fluorescence (Φ = 0.007), which is ascribe to the suppressed ICT
process induced by C=S group and enhanced photo-induced electron transfer (PET) effect of the sulfur atom and phenyl moiety. As expected, the fluorescence intensity at
518 nm increased significantly after adding Cys (Φ1 = 0.237 and Φ2 = 0.106), and
achieved maximum within 25 minutes (Fig. 1). Accordingly, the fluorescence intensity was recorded within 30 minutes. In addition, the absorption spectra of probe
GT-Cys were recorded in the presence and absence of Cys (Fig. S1). As expected, the probe GT-Cys was stable under different conditions, such as wide physiological pH