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).
Autophagy is also induced by the formation and collection of mis-folded proteins in the ER, which causes ER-stress within the cell. Limited autophagy of the cell allows the generation of ATP from digested organelles leading to its ultimate survival. Whilst prolonged adverse conditions results in the death of the cell by the autophagic process.
prior to macroautophagy induced ER phagy and the accumulation of mis-folded proteins within the ER, inclusion bodies form when the ubiquitin-proteasome machinery is overwhelmed by cytostolic protein aggregates to form transient structures termed Aggresome-like Induced structures or ALIS. The formation of ALIS has been shown to require signalling via mTOR and is thus independent of autophagy with ALIS being cleared by lysosomes alone. The binding of LPS or pathogens to TLR receptors not only causes NF-KB acitvation with mTOR signalling but up-regulation of p62 to form ALIS. Aggresomal responses are thought togive rise to Lewy bodies anf hyaline inclusion bodies observed in amyotrophic lateral sclerosis (ALS). Aggresosmes can be detected by use of the Proteostat Aggresome detection kit from Enzo Life Sciences which uses a red fluorescent molecular rotor dye which specifically detects denatured protein cargo within aggresomes and ALISs. Flow cytometry can used as well as fluorescent microscopy by excitation with blue laser and collection at 610nm. Protease inhibitor MG-132 can be used as a positive control for aggresome formation, see figure.
Model of Aggresome and ALIS formation
From:-Protein aggregation and degradation mechanisms in
neurodegenerative diseases by M Takalo, A Salminen, H Soininen, M Hiltunen, A Haapasalo, Am J Neurodegener Dis 2013;2(1):1-14
The ER is responsible for the folding and then delivery of proteins via the secretory pathway to a functional site. Misfolded proteins accumulate in the lumen of the ER due to high protein folding demand on the ER especially during autophagy. Only properly folded proteins are secreted with maintenance of the plasma membrane structure and ER folding capacity being under ER homeostatic control. Once a threshold of mis-folded protein accumulation has been reached, a signal activates the ER to nucleus signalling pathway or the unfolded protein response (UPR) causing ER chaperone proteins to be synthesised, which refold the mis folded proteins and translocate these proteins to the cytoplasm for degradation by the proteasome. This process results in an increase of the ER capacity to fold proteins and maintain ER homeostasis. This increase in ER capacity is physically achieved by an increase in size of the ER at early stages of autophagy, see figure.
If the protein folding demand continues to increase, this will ultimately result in the phagy of the ER itself, which can be caused by ER stress (induced in vitro by tunicamycin and dithiothreitol, DTT), and also multiple mechanisms that induce autophagy e.g.drugs such as rapamycin and nutrient starvation, see figure.
Schematic of ER Phagy
- ER Tracker Green Protocol
- MitoTracker Green Protocol
- Aggresome Protocol