Related Experiment Video
Updated: May 28, 2026
11:39
Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry
Published on: July 21, 2017
"Autophagic flux" in normal mouse tissues: focus on endogenous LC3A processing.
Christos E Zois1, Alexandra Giatromanolaki, Efthimios Sivridis
1Department of Radiotherapy-Oncology, Democritus University of Thrace Medical School, University General Hospital of Alexandroupolis, Alexandroupolis, Greece.
Autophagy
|October 15, 2011
Summary
Autophagy flux can be reliably measured using LC3A processing in mouse tissues. This study confirms LC3A as a key marker, unlike LC3B, for tracking autophagic activity under various stress conditions.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Autophagy is a crucial cellular process for recycling damaged components.
- LC3B is the established marker for autophagy, but its II form's correlation with autophagic membranes is debated.
- The utility of LC3A as an autophagic flux marker remains unclear.
Purpose of the Study:
- To investigate the role of LC3A processing as an endogenous marker for autophagic flux.
- To compare the responsiveness of LC3A and LC3B in mouse tissues under stress.
- To validate antibody specificity for LC3A and LC3B in immunoblotting.
Main Methods:
- Tested specificity of rabbit polyclonal antibodies against human recombinant LC3A and LC3B proteins.
- Measured autophagic flux in mouse liver, lung, kidney, and heart using chloroquine, nutrient starvation, and ionizing radiation.
- Utilized immunoblotting on fractionated (soluble vs. membrane) samples.
Main Results:
- LC3A protein exhibited distinct LC3A-I and LC3A-II conversion patterns across tested mouse tissues under stress.
- LC3B protein did not consistently follow the same pattern as LC3A under the same stressor conditions.
- Antibody specificity was confirmed against recombinant proteins.
Conclusions:
- Endogenous LC3A processing serves as a significant marker for autophagy flux in mouse models.
- Fractionated samples are essential for accurate discrimination of LC3-I and LC3-II in immunoblotting.
- Careful validation of antibody specificity is critical when studying LC3A and LC3B in vivo due to non-overlapping expression and responsiveness.
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,...
01:18Autophagic Cell Death
Christian de Duve discovered “autophagy,” a process in which cellular components are engulfed by membrane-bound organelles called autophagosomes. The autophagosomes then fuse with lysosomes to digest the enclosed contents. Autophagy is generally activated in cells to prevent cell death. However, cell death is triggered when the damage is beyond repair.
Autophagy and Apoptosis
Autophagy can activate apoptosis. In normal conditions, the autophagy activating protein Beclin-1 and pro-apoptotic...
Autophagy and Apoptosis
Autophagy can activate apoptosis. In normal conditions, the autophagy activating protein Beclin-1 and pro-apoptotic...
00:58Recycling Endosomes and Transcytosis
The recycling endosome, also known as the endosomal recycling compartment (ERC), is a part of the slow-recycling process of the endocytic pathway. Molecules internalized through receptor-mediated endocytosis are either degraded in the lysosomes or are recycled to the plasma membrane through the fast- or slow-recycling route.
The recycling endosome is not a single organelle but an extensively tubulated network of recycling pathways. It functions in storing molecules or transporting them across...
The recycling endosome is not a single organelle but an extensively tubulated network of recycling pathways. It functions in storing molecules or transporting them across...
01:22Lysosomal Hydrolases
Lysosomes are the site for the degradation of macromolecules and biological polymers released during membrane trafficking events such as secretory, endocytic, autophagic, and phagocytic pathways. The membrane-enclosed area of the lysosome, called the lumen, contains hydrolytic enzymes active in an acidic environment. These acid hydrolases are functional at a pH between 4.5 and 5 and are involved in cellular processes such as cell signaling, energy metabolism, restoration of the plasma membrane,...
01:28Maturation of Endosomes
The early endosome containing internalized molecules matures through transformations in its location, morphology, intraluminal pH, and membrane protein composition. Together, these changes result in a more acidic late endosome that contains multiple intraluminal vesicles; therefore, the late endosome is also called a multivesicular body (MVB).
Changes in location
The maturing endosome moves along microtubules from the periphery of the cell towards the perinuclear region. This movement of the...
Changes in location
The maturing endosome moves along microtubules from the periphery of the cell towards the perinuclear region. This movement of the...