Real-Time Super-Resolution Tracking of mtDNA Remodeling and Inflammatory Release with a Selective Fluorescent Probe

Area of Science:

• Cell Biology
• Molecular Biology
• Biophysics

Background:

• Mitochondrial DNA (mtDNA) plays a crucial role in cellular function and homeostasis.
• Tracking mtDNA dynamics in live cells is essential for understanding development, metabolism, and disease.
• Existing methods for live-cell mtDNA imaging are limited by probe selectivity and photostability.

Purpose of the Study:

• To develop a novel, photostable fluorescent probe for super-resolution tracking of mtDNA dynamics in live cells.
• To investigate the spatiotemporal release of mtDNA under various cellular conditions.
• To establish a versatile platform for advanced mtDNA imaging and analysis.

Main Methods:

• Rational engineering of a wash-free fluorescent probe, mtNARed.
• Stimulated emission depletion (STED) microscopy for super-resolution imaging.
• Fluorescence-lifetime imaging microscopy (FLIM) for complementary readouts.
• Imaging across diverse mammalian cell types, including sperm cells.

Main Results:

• mtNARed exhibits a large Stokes-shift and high photostability, enabling long-term live-cell imaging.
• The probe precisely localizes to mitochondrial nucleoids, distinguishing mtDNA from nuclear DNA.
• Super-resolution imaging revealed progressive mtDNA release under mitochondrial stress and inflammation.
• mtDNA release correlated with mitochondrial depolarization, membrane disintegration, and immune activation.

Conclusions:

• mtNARed provides a robust and versatile platform for advanced mtDNA imaging.
• This probe facilitates the study of mitochondrial genome dynamics, maintenance, and signaling.
• The findings open new avenues for dissecting mitochondrial roles in physiological and pathological states.