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Updated: May 29, 2026
09:34
The Lactate Dehydrogenase Sequestration Assay — A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells
Published on: July 27, 2018
A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery
Hiroyuki Katayama1, Takako Kogure, Noboru Mizushima
1Life Function and Dynamics, ERATO, JST, Bunkyo-ku, Tokyo 113-8519, Japan.
Chemistry & Biology
|August 27, 2011
Summary
Researchers developed a new method to quantify autophagy flux by tracking Keima protein accumulation in lysosomes. This technique successfully identified an autophagy pathway in cells lacking Atg5, where other methods failed.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Autophagy is a crucial cellular process for degrading damaged components.
- Monitoring the complete flux of autophagy, especially lysosomal fusion, remains challenging.
- Existing methods like LC3-based probes have limitations in certain cellular contexts.
Purpose of the Study:
- To develop a sensitive and quantitative technique for monitoring autophagy flux, focusing on lysosomal membrane fusion.
- To characterize autophagy pathways in cells where conventional probes are ineffective.
- To visualize and quantify selective autophagy, such as mitophagy.
Main Methods:
- Utilized Keima, a pH-sensitive fluorescent protein, to track accumulation within lysosomes.
- Employed time-lapse imaging to monitor Keima fluorescence and co-localized markers.
- Developed a quantitative readout based on cumulative fluorescent signals.
- Applied the technique to Atg5-deficient cells and mitophagy studies.
Main Results:
- Demonstrated Keima accumulation as a reliable indicator of autophagy flux and lysosomal fusion.
- Successfully characterized an autophagy pathway in Atg5-deficient cells.
- Showcased the ability to visualize Keima with other fluorophores due to its spectral properties.
- Simultaneously observed Parkin recruitment and mitochondrial degradation during mitophagy.
Conclusions:
- The Keima-based technique provides a sensitive and quantitative method to monitor autophagy flux.
- This approach overcomes limitations of conventional probes in specific cellular models.
- The technique is versatile for studying various forms of autophagy, including mitophagy.
Related Concept Videos
01:36Delivery Pathways to the Lysosome
Eukaryotic cells use different mechanisms to eliminate toxic waste obsolete and worn-out substances. Lysosomes play a pivotal role in this, and hence, these substances are carried to the lysosome from other parts of the cell and extracellular space through different pathways. The most elaborately studied pathways to the lysosome are the endocytic pathways.
Endocytosis
In endocytosis, the cell membrane takes up macromolecules and particles from the surrounding medium. Clathrin-mediated...
Endocytosis
In endocytosis, the cell membrane takes up macromolecules and particles from the surrounding medium. Clathrin-mediated...
01:27Autophagy
Autophagy is a self-digesting process by which a cell protects itself from threats both within and outside the cell, ranging from abnormal proteins to invading bacteria. In this process, obsolete components of the cell and invading microbes are degraded by hydrolytic enzymes active in an acidic environment of the lysosomal lumen.
An autophagic pathway consists of a series of signaling events activated in response to diverse stress and physiological conditions such as food deprivation,...
An autophagic pathway consists of a series of signaling events activated in response to diverse stress and physiological conditions such as food deprivation,...