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Published on: May 4, 2016
Piperazine-Based Mitochondria-Immobilized pH Fluorescent Probe for Imaging Endogenous ONOO- and Real-Time Tracking of
Minglu Li1, Yue Huang1, Shengmei Song2
1College of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, P. R. China.
Abstract:
ONOO- is mainly produced in mitochondria, and dysfunctional and damaged mitochondria are degraded in lysosomes through autophagy, so it is important to synthesize a single probe for dual detection of ONOO- and mitophagy. Unfortunately, mitochondria-immobilized fluorescent probes for dual detection of ONOO- and mitophagy have not yet been developed. Hence, we first reported a piperazine-based mitochondria-immobilized red-emitting fluorescent probe (PMR), which not only can detect ONOO- but also could be used to image cellular mitophagy by the pH variations because of the protonation of the piperazine moiety. PMR was designed and prepared by introducing a piperazine ring as the pH response group, a lipophilic cation as the targeting mitochondria moiety, and benzyl chloride for immobilizing mitochondrial proteins through thiol groups. PMR displayed an enhanced fluorescence response at 640 nm through mitochondrial acidification. Using these advantages of PMR, which was successfully used for visualizing the mitophagy process induced by rapamycin or starvation, and chloroquine can inhibit rapamycin-induced mitophagy and prevent the fusion of autophagosomes and lysosomes. PMR also showed good sensitivity with a detection limit of 23 nM to ONOO-, which was successfully applied in imaging exogenous/endogenous ONOO-. Combining the above design, PMR may be used to study the detailed function of the mitophagy and ONOO--associated physiological and pathological processes.
Insights
Researchers developed a novel red-emitting fluorescent probe (PMR) for simultaneously detecting peroxynitrite (ONOO-) and mitophagy in mitochondria. This probe enables visualization of mitophagy and ONOO- related processes, aiding in understanding mitochondrial dysfunction.
Area of Science:
- Mitochondrial biology
- Cellular imaging
- Biochemistry
Background:
- Mitochondria produce peroxynitrite (ONOO-), a reactive nitrogen species implicated in cellular damage.
- Mitophagy, the degradation of damaged mitochondria via autophagy, is crucial for cellular health.
- Simultaneous detection of ONOO- and mitophagy is needed to understand their interplay, but suitable probes are lacking.
Purpose of the Study:
- To develop and characterize a novel mitochondria-targeted fluorescent probe for dual detection of ONOO- and mitophagy.
- To investigate the probe's ability to visualize mitophagy and ONOO- in live cells.
- To assess the probe's potential in studying ONOO--associated physiological and pathological processes.
Main Methods:
- Synthesis and characterization of a piperazine-based, mitochondria-immobilized red-emitting fluorescent probe (PMR).
- Utilizing PMR's pH-sensitive piperazine moiety for mitophagy imaging and fluorescence response to mitochondrial acidification.
- Applying PMR to visualize mitophagy induced by rapamycin/starvation and its inhibition by chloroquine.
- Assessing PMR's sensitivity for detecting exogenous and endogenous ONOO-.
Main Results:
- PMR was successfully synthesized and demonstrated mitochondria-targeting and immobilization capabilities.
- PMR exhibited enhanced red fluorescence (640 nm) upon mitochondrial acidification, enabling mitophagy visualization.
- PMR successfully imaged mitophagy induction and inhibition, and detected ONOO- with a detection limit of 23 nM.
- The probe effectively visualized both exogenous and endogenous ONOO- in cellular models.
Conclusions:
- A novel piperazine-based fluorescent probe (PMR) was developed for simultaneous dual detection of ONOO- and mitophagy in mitochondria.
- PMR facilitates real-time imaging of mitophagy and ONOO- levels, offering insights into mitochondrial dynamics.
- This probe holds promise for investigating the roles of mitophagy and ONOO- in various physiological and pathological conditions.
