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Visualizing Mitophagy with Fluorescent Dyes for Mitochondria and Lysosome
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Fluorogenic Substrates for Visualizing Acidic Organelle Enzyme Activities.

Fiona Karen Harlan1, Jason Scott Lusk1, Breanna Michelle Mohr2

  • 1Research and Development, Marker Gene Technologies, Inc., Eugene, OR, United States of America.

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|May 27, 2016
PubMed
Summary
This summary is machine-generated.

New fluorogenic probes effectively label lysosomes and monitor their metabolic activity in live cells. These probes show promise for diagnosing lysosomal storage diseases and developing new therapies.

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Area of Science:

  • Cell Biology
  • Biochemistry
  • Molecular Biology

Background:

  • Lysosomes are vital acidic organelles in mammalian cells, regulating processes like metabolism, signaling, and pathogen defense.
  • Lysosomal dysfunction is linked to severe neurological disorders, including Parkinson's, Alzheimer's, and Lysosomal Storage Diseases (LSDs).
  • Accurate monitoring of lysosomal function and enzyme activity is crucial for understanding these diseases and developing treatments.

Purpose of the Study:

  • To develop novel fluorogenic lysosomal staining probes for live-cell imaging.
  • To assess the probes' ability to monitor lysosomal metabolic activity and specific enzyme functions.
  • To evaluate the probes' utility in diagnosing LSDs by analyzing lysosomal enzyme levels in patient-derived cells.

Main Methods:

  • Synthesis of targeted fluorogenic substrates utilizing low pKa fluorescent dyes and enzyme-cleavable moieties.
  • Live-cell staining of patient-derived cells (Metachromatic Leukodystrophy, Krabbe, Gaucher Diseases) and control cells.
  • Comparison of probe localization with established lysosomal markers (LysoTracker® Red, anti-LAMP1 Antibody).
  • Assessment of metabolic activity by inhibiting cellular metabolism with chloroquine.
  • Quantification of staining intensity in relation to lysosomal enzyme levels in diseased versus healthy cells.

Main Results:

  • The synthesized probes successfully localized to lysosomes in various human cell types, confirmed by LysoTracker® and LAMP1 staining.
  • Probes demonstrated the capacity to monitor lysosomal metabolic activity, with staining intensity decreasing upon metabolic inhibition.
  • Staining intensity directly correlated with endogenous lysosomal enzyme levels, showing reduced signal in cells with specific enzyme deficiencies (Gaucher, Krabbe diseases).
  • Substrates designed for Gaucher and Krabbe diseases exhibited diminished staining in corresponding patient cell lines.

Conclusions:

  • Novel lysosome-targeted fluorogenic substrates enable effective live-cell labeling and functional assessment of lysosomes.
  • These probes can accurately reflect lysosomal enzyme activity and metabolic status in real-time.
  • The developed substrates offer a valuable tool for research, diagnostics of lysosomal storage disorders, and monitoring therapeutic efficacy in drug development.