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Autophagy

Autophagy - Blizard Institute

Autophagy

The process of autophagy is a cell survival mechanism that occurs when the cell is under stress, via external environmental pressures, including the lack of nutrients, or via the internal microenvironment of the cell, including the replacement of old and defective organelles such as mitochondria & the Endoplasmic Reticulum (ER).

Annexin-V - Cell membrane changes

In normal cells, phosphatidylserine (PS) residues are found in the inner membrane of the cytoplasmic membrane.  During apoptosis, the PS residues are translocated in the membrane and are externalized.  In general, this is not an early event in autophagy and the the role of PS externalization is unclear in autophagy. Interestingly the phospholipid, phosphatidyl- ethanolamine (PE) plays a crucial role in the formation of the autophagosome during the induction phase of the autophagic response in that it lipidates the microtubule LC3 in the double membrane of the autophagosome to form LC3B which is used as a marker for autophagosomes and hence the detection of autophagic cells.

Role of microtubule protein LC3 in autophagy

There are numerous methods of inducing autophagy including serum starvation and rapamycin which both inhibit mTOR signalling. Chloroquine interestingly induces the formation of autophagosomes but blocks the formation of autophagolysosomes and hence ultimately is an inhibitor of autophagy. The ER-ATPase inhibitor, Thapsigargin has also been reported to induce autophagy of the ER.

There are numerous methods of inducing autophagy including nutrient starvation and rapamycin which both inhibit mTOR signalling. The lack of nutrients such as serum and amino acids in the cell environment has been known to cause cell cycle arrest as well as autophagy and indeed the immunosuppressive antibiotic, rapamycin is a known to target phosphatidylinositol kinase-related kinase mTOR which combines the nutrient and mitogen signals that regulate cell cycle as well as induction of autophagy by inhibition of mTOR. Lack of serum growth factors, essential amino acids (EAA) as well as glutamine (Gln a necessary supplement for cell lines) and rapamycin has been shown to cause G1 cell cycle arrest at the restriction point (R) which is upstream of the checkpoint for EAA which is upstream of the Gln checkpoint with the rapamycin checkpoint being downstream of both of these check point just above S phase. Flow cytometry has previously been employed not only to measure LC3B but combine this with cell cycle analysis to demonstrate cell cycle arrest by rapamycin and various nutrients.

Role of electron microscopy in the characterization of autophagy

Autophagy was originally characterized in 1963 by De Duve with electron microscopy studies showing the presence of double membrane bound structures termed auto-phagosomes and single membrane vesicles terming autolysosomes or autophagolysosomes, see figure.

Role of lysosomes in autophagy

There are numerous methods of inducing autophagy including serum and total nutrient starvation and rapamycin which both inhibit mTOR signalling. Chloroquine interestingly induces the formation of autophagosomes but blocks the formation of autophagolysosomes and hence ultimately is an inhibitor of autophagy. The ER-ATPase inhibitor, Thapsigargin has also been reported to induce autophagy of the ER. Chaperone Mediated Autophagy or CMA in which cytosolic proteins are sequestered to lysosomes via a protein chaperone e.g. cytosolic protein hsc70 and are then degraded after binding to lysosomal membrane protein, LAMP-2.

Plasma membrane changes

In normal cells, phosphatidylserine (PS) and phosphatidyl-ethanolamine (PE) residues are found in the inner membrane of the cytoplasmic membrane.  During apoptosis, the PS and PE residues are translocated in the membrane and are externalised resulting in a change in surface charge of the outer leaflet of the plasma membrane.  In general, though not always, this is an intermediate event in apoptosis and is thought to be a signal to neighbouring cells that a cell is ready to be phagocytosed.  Annexin-V is a specific PS-binding protein that can be used to detect apoptotic cells by binding to PS. In general, this is not an early event in autophagy and the the role of PS externalisation is unclear in autophagy.  Interestingly the phospholipid, phosphatidyl- ethanolamine (PE) plays a crucial role in the formation of the autophagosome during the induction phase of the autophagic response in that it lipidates the microtubule LC3 in the double membrane of the autophagosome to form LC3B which is used as a marker for autophagosomes and hence the detection of autophagic cells.

Endoplasmic Reticulum phagy

The process of autophagy is a cell survival mechanism that occurs when the cell is under stress, via external environmental pressures, including the lack of nutrients, or via the internal microenvironment of the cell, including the replacement of old and defective organelles such as mitochondria.

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