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.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

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

The title of the Nobel Lecture of George Palade (1 August, p. 347) should have been "Intracellular aspects of the process of protein secretion."

https://science.sciencemag.org/content/sci/189/4200/347.full.pdf

Intracellular aspects of the process of protein synthesis

This lecture summarizes the knowledge in a field dubbed autopharmacology: the secretion by a body cell of a substance prompted by an internal stimulus. The majority of the work reported on here was with the adrenal chromaffin cell. Topics covered describing and proving the release of secretion into the chromaffin cells extracellular environment are means of intracellular recording from chromaffin cells and the effect of acetylcholine on membrane potential and consequences of exposing the cell to acetylcholine (altered membrane chemistry increased membrane permeability entry of sodium and calcium ions and depolarization). Also postulated are 2 possible mechanisms of catecholamine secretion (both involve release directly from chromaffin granules to the cell exterior through frank rupture of the adherent membranes of granule and cell i.e. exocytosis or through some greatly increased permeability at the adhesion site). The lecture then focuses on calcium and stumulus secretion coupling in other systems including the exocrine glands neurones and neurosecretory cells the mast cell and polymorphonuclear leukocytes. In a concluding discussion on the role of calcium in secretory mechanisms the presence of prostaglandin in effluent from adrenal glands exposed to acetylcholine is mentioned as evidence of the secretory mechanism being a chemical reaction.

Stimulus-secretion coupling: the concept and clues from chromaffin and other cells.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

Neutrophilic leukocytes (PMN) and their precursors from normal human marrow and blood were examined by histochemical staining and by electron microscopy and cytochemistry in order to determine the origin and nature of their cytoplasmic granules. Human neutrophils contain two basic types of granules, azurophils and specifics, which differ in morphology, contents, and time of origin. Azurophils are large and may be spherical or ellipsoid, the latter with a crystalline inclusion. They are produced in the first secretory stage (promyelocyte), contain peroxidase and various lysosomal enzymes, and thus correspond to modified primary lysosomes. Specifics are smaller, may be spherical or elongated, and are formed during a later secretory stage (myelocyte). They lack lysosomal enzymes and contain alkaline phosphatase and basic protein; their contents remain largely undetermined. Specifics outnumber azurophils in the mature PMN because of reduction in numbers of azurophils per cell by cell division in the myelocyte stage. The findings indicate that the situation is basically the same as described previously in the rabbit, insofar as the origin, enzymic activity, and persistence in the mature cell of the two types (azurophil and specific) of granules are concerned. The main difference between PMN of the two species is in the morphology (size, shape, and density) of the granules, especially the azurophils.

/pdf/the-development-of-neutrophilic-polymorphonuclear-leukocytes-4nwo3ilzo6.pdf

The development of neutrophilic polymorphonuclear leukocytes in human bone marrow origin and content of azurophil and specific granules

The nature and content of lytic bodies and the localization of acid phosphatase (AcPase) activity were investigated in mammotrophic hormone-producing cells (MT) from rat anterior pituitary glands. MT were examined from lactating rats in which secretion of MTH(1) was high and from postlactating rats in which MTH secretion was suppressed by removing the suckling young. MT from lactating animals contained abundant stacks of rough-surfaced ER, a large Golgi complex with many forming secretory granules, and a few lytic bodies, primarily multivesicular bodies and dense bodies. MT from postlactating animals, sacrificed at selected intervals up to 96 hr after separation from their suckling young, showed (a) progressive involution of the protein synthetic apparatus with sequestration of ER and ribosomes in autophagic vacuoles, and (b) incorporation of secretory granules into multivesicular and dense bodies. The content of mature granules typically was incorporated into dense bodies whereas that of immature granules found its way preferentially into multivesicular bodies. The secretory granules and cytoplasmic constituents segregated within lytic bodies were progressively degraded over a period of 24 to 72 hr to yield a common residual body, the vacuolated dense body. In MT from lactating animals, AcPase reaction product was found in lytic bodies, and in several other sites not usually considered to be lysosomal in nature, i.e., inner Golgi cisterna and associated vesicles, and around most of the immature, and some of the mature secretory granules. In MT from postlactating animals, AcPase was concentrated in lytic bodies; reaction product and incorporated secretory granules were frequently recognizable within the same multivesicular or dense body which could therefore be identified as "autolysosomes" connected with the digestion of endogenous materials. Several possible explanations for the occurrence of AcPase in nonlysosomal sites are discussed. From the findings it is concluded that, in secretory cells, lysosomes function in the regulation of the secretory process by providing a mechanism which takes care of overproduction of secretory products.

/pdf/lysosome-function-in-the-regulation-of-the-secretory-process-23amunpzvt.pdf

Robert E. Smith

Papers

Lysosome function in the regulation of the secretory process in cells of the anterior pituitary gland.