Visualizing Cardiolipin In Situ with HKCL-1M, a Highly Selective and Sensitive Fluorescent Probe

Abstract

Reliable probing of cardiolipin (CL) content in dynamic cellular milieux presents significant challenges and great opportunities for understanding mitochondria-related diseases, including cancer, neurodegeneration, and diabetes mellitus. In intact respiring cells, selectivity and sensitivity for CL detection are technically demanding due to structural similarities among phospholipids and compartmental secludedness of the inner mitochondrial membrane. Here, we report a novel “turn-on” fluorescent probe HKCL-1M for detecting CL in situ. HKCL-1M displays outstanding sensitivity and selectivity toward CL through specific noncovalent interactions. In live-cell imaging, its hydrolyzed product HKCL-1 efficiently retained itself in intact cells independent of mitochondrial membrane potential (Δψm). The probe robustly co-localizes with mitochondria and outperforms 10-N-nonyl acridine orange (NAO) and Δψm-dependent dyes with superior photostability and negligible phototoxicity. Our work thus opens up new opportunities for studying mitochondrial biology through efficient and reliable visualization of CL in situ.

Lighting Up Nucleolus To Report Mitochondria Damage Using a Mitochondria-to-Nucleolus Migration Probe

“…The syntheses of SYN and BYD were achieved in one step via the classic Knoevenagel condensation reaction (Scheme S1). The chemical structures of these molecules were completely characterized using 1 H NMR, 13 C NMR, and HRMS (Figures S14−S19) to confirm their structure and purity.…”

“…Materials and instrumentation; synthesis of probes and characterization ( 1 H NMR, 13 C NMR, and HRMS); photophysical properties; HOMO/LUMO orbital calculation; molecular docking; CD spectra; MTT assay for cell cytotoxicity; cell culture and staining methods; CLSM images; docked free energies (PDF)…”

“…Currently, several methods have been explored for observing mitochondria, such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), electron probe X-ray microanalyzer (EPMA), etc . − However, these methods do not allow for live-cell imaging or the real-time monitoring of mitochondria. Fluorescence microscopy is a valuable tool for real-time visualization of bioactive molecules in situ due to its noninvasive nature − using protein markers or fluorescence in situ hybridization (FISH) . However, these techniques often require expensive protein markers or gene modification.…”

Light-Driven Mitochondrion-to-Nucleus DNA Cascade Fluorescence Imaging and Enhanced Cancer Cell Photoablation

“…Fluorescence imaging techniques are widely used to visualize biological structures, track physiological and pathological processes, and diagnose diseases in biological systems owing to their high spatial resolution, ability to allow real-time visualization, and noninvasive nature. − To date, a few fluorescent probes have been developed to accumulate in mitochondria, relying on the high mitochondrial membrane potential (MMP), or to visualize the nucleus by targeting relevant proteins. − Additionally, numerous nucleic acid-based fluorescent probes have been developed to target the mitochondrion or the nucleus. − However, none of them can simultaneously detect nucleic acids in mitochondria and nuclei in a cascade mode. Because the nucleic acid changes in the mitochondrion and the nucleus are closely related to cell death and cancer, it is crucial to rationally design a nucleic acid-based fluorescent molecule for mitochondrion and nucleus imaging or treatment.…”