In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.

For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

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Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In the cell, as in vitro, the final conformation of a protein is determined by its amino-acid sequence. But whereas some isolated proteins can be denatured and refolded in vitro in the absence of other macromolecular cellular components, folding and assembly of polypeptides in vivo involves other proteins, many of which belong to families that have been highly conserved during evolution.

Protein folding in the cell.

Significance: Autophagy is a highly conserved eukaryotic cellular recycling process. Through the degradation of cytoplasmic organelles, proteins, and macromolecules, and the recycling of the breakdown products, autophagy plays important roles in cell survival and maintenance. Accordingly, dysfunction of this process contributes to the pathologies of many human diseases. Recent Advances: Extensive research is currently being done to better understand the process of autophagy. In this review, we describe current knowledge of the morphology, molecular mechanism, and regulation of mammalian autophagy. Critical Issues: At the mechanistic and regulatory levels, there are still many unanswered questions and points of confusion that have yet to be resolved. Future Directions: Through further research, a more complete and accurate picture of the molecular mechanism and regulation of autophagy will not only strengthen our understanding of this significant cellular process, but will aid in the development o...

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An Overview of Autophagy: Morphology, Mechanism, and Regulation

Heat shock proteins.

For a long time, lysosomes were considered merely to be cellular 'incinerators' involved in the degradation and recycling of cellular waste. However, now there is compelling evidence indicating that lysosomes have a much broader function and that they are involved in fundamental processes such as secretion, plasma membrane repair, signalling and energy metabolism. Furthermore, the essential role of lysosomes in autophagic pathways puts these organelles at the crossroads of several cellular processes, with significant implications for health and disease. The identification of a master regulator, transcription factor EB (TFEB), that regulates lysosomal biogenesis and autophagy has revealed how the lysosome adapts to environmental cues, such as starvation, and targeting TFEB may provide a novel therapeutic strategy for modulating lysosomal function in human disease.

Signals from the lysosome: a control centre for cellular clearance and energy metabolism.

A 73-kilodalton (kD) intracellular protein was found to bind to peptide regions that target intracellular proteins for lysosomal degradation in response to serum withdrawal. This protein cross-reacted with a monoclonal antibody raised to a member of the 70-kD heat shock protein (hsp70) family, and sequences of two internal peptides of the 73-kD protein confirm that it is a member of this family. In response to serum withdrawal, the intracellular concentration of the 73-kD protein increased severalfold. In the presence of adenosine 5'-triphosphate (ATP) and MgCl2, the 73-kD protein enhanced protein degradation in two different cell-free assays for lysosomal proteolysis.

https://science.sciencemag.org/content/sci/246/4928/382.full.pdf

A role for a 70-kilodalton heat shock protein in lysosomal degradation of intracellular proteins

Protein and peptide binding and stimulation of in vitro lysosomal proteolysis by the 73-kDa heat shock cognate protein.

In response to serum withdrawal, when overall rates of proteolysis increase in cultured fibroblasts, proteins containing peptide regions similar to Lys-Phe-Gln-Arg-Gln (KFERQ) are targeted to lysosomes for degradation, and the intracellular concentrations of these proteins decline [Chiang & Dice (1988) J. Biol. Chem. 263, 6797-6805]. To test whether such proteins are also selectively depleted in mammalian tissues in vivo, we have used affinity-purified polyclonal antibodies to KFERQ to detect proteins containing such sequences in tissues of fed and fasted rats. Immunoreactive cytosolic proteins were partially depleted from liver and heart of fasted rats, but the time course differed for these two tissues. Immunoreactive proteins in liver were lost during days 2 and 3 of fasting, whereas such proteins in heart were depleted within day 1 of fasting. In the same fasted rats, levels of immunoreactive cytosolic proteins did not change in two skeletal muscles, the dark soleus and the pale extensor digitorum longus. Immunoreactive proteins in a myofibrillar fraction were also partially depleted in heart, but not in skeletal muscles, of fasted rats. The most likely explanation for these results is that the protein loss in different tissues upon fasting results from selective activation of different proteolytic pathways. The increased proteolysis in liver and heart of fasted animals includes activation of the KFERQ-selective lysosomal pathway, whereas increased proteolysis in skeletal muscle does not.

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Proteins containing peptide sequences related to Lys-Phe-Glu-Arg-Gln are selectively depleted in liver and heart, but not skeletal muscle, of fasted rats.

Hui Ling Chiang

Papers

A role for a 70-kilodalton heat shock protein in lysosomal degradation of intracellular proteins

Protein and peptide binding and stimulation of in vitro lysosomal proteolysis by the 73-kDa heat shock cognate protein.

Proteins containing peptide sequences related to Lys-Phe-Glu-Arg-Gln are selectively depleted in liver and heart, but not skeletal muscle, of fasted rats.