Autophagy is an evolutionarily conserved process essential for survival, differentiation, development, and homeostasis. However, its involvement in several diseases has been established for many years. Read our blog below to learn more about autophagy and some common approaches to detect it.

 

Autophagy

Autophagy is a physiological process responsible for the degradation of cytoplasmic components within lysosomes. It involves the formation of double-membraned vesicles called autophagosomes that encapsulate cytoplasmic contents and deliver the cargo to the lysosome for destruction. It increases in response to cell stress such as deprivation of nutrients or growth factors. Autophagy plays a crucial role in turnover of long-lived, misfolded or aggregated proteins, damaged or redundant organelles, as well as in elimination of intracellular pathogens.

Fun fact- Autophagy

Although widely viewed as an essential process to maintain cellular homeostasis and functions, dysregulation in autophagy can have dire consequences. Its linkage to cancer, muscular and neurodegenerative diseases has generated a lot of interest in investigating autophagic pathways. Below we have discussed one of the main biomarkers used to monitor autophagy.

 

LC3B- An important Autophagy Biomarker

LC3B, a sub-unit of microtuble-associated protein LC3 (light chain 3), is one of the most reliable and widely used autophagy biomarkers. Along with other associated molecules, it helps guide and regulate autophagosome assembly and formation. After synthesis, LC3B is cleaved to expose a C-terminal glycine, representing the inactive cytosolic form LC3B-I. During autophagy, the C-terminus covalently links to autophagosomal vesicle membranes and is called LC3B-II. The conversion of LC3B-I to LC3B-II is therefore usually associated with the formation of autophagosomes. As autophagosomes fuse with lysosomes to form autolysosomes, LC3B-II in the autolysosomal lumen gets degraded. This lysosomal turnover of the autophagosomal marker LC3B-II reflects starvation-induced autophagic activity. Learn more about LC3 by following the link to this review.

Autophagic flux

Simplistic representation of autophagic flux, including LC3 processing

NanoBit® – A simple to perform, homogenous assay

There are various approaches available to measure LC3B expression and detect autophagy. As shown in this poster, we have validated NanoBiT® Luciferase technology (Promega) against standard technologies such as Western blotting, AlphaLISA and High Content Imaging. The NanoBiT® system is composed of two subunits, Large BiT (LgBiT; 17.6 kDa) and Small BiT (SmBiT; 11 amino acid peptide) and can be used for intracellular detection of protein: protein interactions (PPI). When two proteins of interest are tagged with these sub-units and then interact, the sub-units come together to form an active enzyme that generates a bright luminescent signal.  LgBiT and SmBiT have been optimised for stability and minimal self-association. Interaction of both the sub-units is reversible, and so the system can be used to detect rapidly dissociating proteins. The proportional signal-to-activity and plate-based format make NanoBiT® ideal for PPI studies, including high-throughput screening. Follow the link to our poster to find out more about our study.

NanoBit®

NanoBiT® system

Are you examining autophagy as a part of your research? Why not get in touch with our experienced team of scientists and take your project to the next stage?

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