Research Techniques for Autophagy
Normally cultured cells have low autophagic properties and are not suitable for observation. Therefore, manual intervention and regulation of autophagy must be performed. The reported tools are:
1, autophagy inducer
(1) Bredeldel A/Thasigargin/ Tunicamycin: mimics endoplasmic reticulum stress.
(2) Carbamazeoine/ L-690, 330/ Lithium Chloride: IMPase (Inositol monop hosphatase)
(3) Earle’s Balanced Salt Solution: Used to create hunger.
(4) N-Acetyl-D-sphingosine (C2-ceramide): Class I PI3K Pathway inhibitor
(5) Rapamycin: mTOR inhibitor
(6) Xestospongin B/C: IP3R blocker
2, autophagy inhibitor
(1) 3-Methyladenine (3-MA): Class Ill PI3Khps34 inhibitor
(2) Bafilomycin A1: Proton pump inhibitor
(3) Hydroxychloroquine (hydroxy chloroquine): Lysosomal|u men alkalizer (lysosomal cavity alkalinizing agent)
In addition to the selection of the above-mentioned tools, it is generally necessary to combine the genetic techniques to intervene in autophagy-related genes: including antisense RNA interference technology (Knockdown), screening of mutant strains, and introduction of foreign genes.
3, autophagy process for observation and detection
After the cells are induced or inhibited, the autophagy process needs to be observed and detected. Common strategies and techniques are:
(1) Observing the formation of autophagosomes
Since autophagosomes belong to the subcellular structure, they are not visible under ordinary light microscope. Therefore, direct observation of autophagosomes should be performed under transmission electron microscopy. Phagophores are characterized by a crescent-shaped or cup-shaped, double-layer or multi-layered film that has a tendency to surround the cytosolic composition. Autophagosomes (AV1) are characterized by a vacuole-like structure of a bilayer or multilayer membrane containing cytosolic components such as mitochondria, endoplasmic reticulum, ribosomes, and the like. Autophagic lysosomes (AV2) are characterized by a monolayer membrane in which the cytosolic component has been degraded. (autophagicvacuole, AV)
(2) Using GFP-LC3 fusion protein to trace autophagy formation under fluorescence microscope
Because the electron microscope takes a long time, it is not conducive to the monitoring of autophagy formation. People have developed this technology by using LC3 to accumulate during autophagy formation. When there is no autophagy, the GFP-LC3 fusion protein is dispersed in the cytoplasm; when autophagy is formed, GFP-LC3 fusion translocates to the autophagosome membrane, and multiple green fluorescent spots are formed under the fluorescence microscope. One spot is equivalent to one self. For phagosomes, the level of autophagy activity can be assessed by counting.
(3) Using Western blo to detect changes in LC3 ratio to evaluate autophagy formation
When autophagy is formed, cytosolic LC3 (ie, LC3-) will amplify a small segment of the polypeptide and transform it into an (autophagosome) membrane type (ie, LC3III). Therefore, the LC3-/ratio can estimate the level of autophagy. High and low.
(Note: LC3 antibodies have a higher affinity for LC3-, which can cause false positives. Methods 2 and 3 need to be combined, taking into account the effects of lysosomal activity.)
(4) Detection of mass degradation of longevity proteins: non-specific
(5) MDC (monodansylcadaverine, monodansyl sulfonamide) staining: including autophagosomes, all acidic vacuoles are stained, so it is non-specific.
(6) CellTracker TM Green staining: mainly used for double staining but it can stain all vacuoles, so it is also non-specific.
4, localization of autophagy-related proteins
When studying autophagy-related proteins, they need to be localized. Since autophagosomes are closely related to lysosomes, mitochondria, endoplasmic reticulum, and Golgi, some tracer proteins are commonly co-localized under fluorescence microscopy for differentiation:
Lamp2: a lysosomal membrane protein that can be used to monitor autophagosome fusion with lysosomes.
Lyso TrackerTM Probe: Available in red or blue to display all acidic vacuoles.
pDsRed2mito: vector, which expresses a fusion protein (red fluorescent protein + mitochondrial matrix localization signal) after transfection and can be used to detect the degree of autophagy of mitochondria (Mitophagy).
Mito trake probe: specifically displays live mitochondria, and fluorescence remains after fixation.
Hsp60: localizes to the mitochondrial matrix and is not released when cells die.
Calreticulin: the endoplasmic reticulum.
[Note: These proteins are all cytosolic proteins. Cells that are scrambled or trypsinized must be permeable-e before immunofluorescence and can be treated with 0.1% SDS. ]