Autophagy Monitoring
Autophagy Monitoring - Related Videos
08:44
Activating Autophagy by Aerobic Exercise in Mice
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Cited by 37 •
2017
Autophagy activation is beneficial in the prevention of a number of diseases. One of the physiological approaches to induce autophagy in vivo is physical exercise. Here we show how to activate autophagy by aerobic exercise and measure autophagy levels in mice.
09:00
Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond
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Cited by 13 •
2013
Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.
12:52
Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions
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Cited by 14 •
2014
To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.
14:08
Study of Protein-protein Interactions in Autophagy Research
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Cited by 3 •
2017
Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.
09:59
Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy
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Cited by 3 •
2013
Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.
FAQs
LC3 conversion alone does not establish that the pathway is completing its task. A rise in LC3 can accompany greater autophagic activity, but it may also reflect reduced lysosomal clearance. Measuring p62 degradation alongside LC3, and examining responses with lysosomal inhibition, helps distinguish increased material delivery from accumulation caused by impaired downstream processing.
Lysosomal inhibitors temporarily block the clearance step, allowing researchers to compare marker levels when degradation is permitted with levels when it is interrupted. An increase that appears under inhibition supports ongoing delivery toward lysosomes, whereas marker accumulation without this comparison may indicate defective clearance. This paired design therefore improves interpretation of autophagic flux.
Fluorescence microscopy, immunoblotting, and reporter systems provide complementary views of pathway activity. Microscopy can track fluorescence-based patterns in cells, immunoblotting measures marker changes such as LC3 conversion or p62 degradation, and reporters provide a system for following pathway behavior. Combining readouts can make conclusions more informative than relying on one measurement.
A useful comparison keeps the readout and experimental logic consistent while varying the condition of interest, such as nutrient stress, drug treatment, or a disease-related state. Marker measurements can then be evaluated with and without lysosomal inhibition. This approach helps determine whether a treatment changes pathway delivery, clearance, or both.
Researchers first apply the relevant condition, then assess LC3 conversion and p62 degradation using fluorescence microscopy, immunoblotting, or a reporter system. Parallel measurements with lysosomal inhibitors provide the comparison needed to interpret clearance. The resulting pattern indicates whether the tested condition is associated with altered autophagic flux rather than simply a change in one marker.
Autophagy monitoring connects cellular pathway measurements with questions about homeostasis and disease-related biology. It can reveal responses to nutrient stress or drug treatment and support studies of cancer, neurodegeneration, infection, metabolism, and therapeutic responses. In each setting, measuring flux helps researchers relate changes in pathway activity to the cellular condition being investigated, rather than treating one marker as a complete result.