Adenovirus Production for Autophagy Flux Detection

Autophagy is an intracellular system that degrades cytosolic proteins and organelles. Autophagy can be divided into three groups: macroautophagy, microautophagy and chaperon-mediated autophagy (CMA). Macroautophagy is most extensively studied and best known among the three pathways. When macroautophagy is induced, an isolation membrane encloses a portion of cytoplasm, forming a characteristic double-membraned organelle called the autophagosome. The autophagosome then fuses with the lysosome to form an autolysosome, then the contents of which are degraded by lysosomal enzymes. Autophagic degradation of cytoplasmic components is essentially non-selective, but can also be selective. Non-selective autophagy is important for starvation adaptation, whereas selective autophagy may be more important for maintaining homeostasis of organelles and cytosolic proteins, although these two categories are not mutually exclusive. Autophagy has also been implicated in pathological conditions including neurodegenerative diseases, cancer, and inflammatory diseases. Modulation of autophagy has become a potential therapeutic target in human diseases.

To monitoring and measuring autophagy, it is necessary to label and visualize the phagophores and autophagosomes. However, few proteins are uniquely associated with autophagic vesicles and their precursors, with only one protein family (including LC3-II) known to label autophagic structures both before and after fusion with the lysosome. LC3 is one of several vertebrate homologues of ATG8. ATG4 is a cysteine protease which cleaves the C-terminus of LC3, exposing a glycine residue. This first cleaved form of LC3 is called LC3-I. A further reaction then occurs involving a complex of ATG proteins that act as an E3-like ligase. This determines the site of LC3 lipidation and assists the transfer of LC3-I to PE to form LC3-II. Currently, detecting LC3 by immunoblotting or immunofluorescence has become a reliable method for monitoring autophagy and autophagy-related processes, including autophagic cell death.

QVirus™ Platform has launched series of adenovirus packaging service of autophagy related biosensors, in which GFP and/or RFP tags are fused at the C-termini of the autophagosome marker LC3, allowing to detect the intensity of autophagy flux in real-time with more accuracy, clarity and intuitiveness. The fluorescent proteins GFP and mRFP have different properties under acidic conditions. Consequently, GFP channels and mRFP channels of the same labeled cells showed different distribution patterns of puncta. The development of the tandem fluorescent mRFP-GFP-LC3 has been widely used in vitro to study the mechanisms regulating the maturation of autophagosomes and the fusion to lysosomes in the degradative process. Due to this pH-dependent quenching of the GFP-LC3 fluorescence, only mRFP-LC3 can be detected in autolysosomes (i.e. these appear red only), whereas autophagosomes can be visualized by both fluorophores (i.e. these appear yellow).