The autophagy community

There are various definitions of community. A definition that I found in one of my dictionaries is the following: “A social, religious, occupational, or other group sharing common characteristics or interests and perceived or perceiving itself as distinct in some respect from the larger society within which it exists.” Thus, I think it is fair to say that there is a worldwide autophagy community. That is, there is a group of researchers (our occupation), whose members share an interest in autophagy (our common characteristic), and that group is distinct from the larger society (I do not want to begin describing the many ways this applies). But do we feel like a community, and do we need a community? I suggest that a community is indeed beneficial, and I propose one mechanism for enhancing the development of the autophagy community.     

A human autophagy interaction network

The original “Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes” has been well received and used by many researchers and authors. I consider these to be very important guidelines that require a consensus among the researchers in the field, because they are used by authors to defend against inappropriate reviewers’ comments, and by reviewers to point out to editors the flaws in research papers. Accordingly, I decided it was time to revise and update the guidelines. After all, the field has expanded substantially, as has the range of model systems being used to analyze autophagy. As a result, the list of authors has similarly increased. In addition, this version of the guidelines is not limited to higher eukaryotes nor to macroautophagy. Here, I explain the approach used to invite authors to participate in the revised guidelines, and briefly demonstrate one aspect of their utility.     

Finding autophagy

To tell the truth, I find it difficult to work when flying, or even when sitting in an airport for an extended period of time. So, typically I take along a book to read. And when I truly cannot concentrate, for example when a flight is considerably delayed, I have even been known to resort to word puzzles. Depending on the type, they do not require much attention (that is, you can pick up right where you left off after you glance at the flight status screen for the twentieth or so time, even though you know nothing has changed), or effort (although you need to use a pen or pencil, not a keyboard), but nonetheless they can keep your mind somewhat occupied. I even rationalize doing them based on the assumption that they are sharpening my observational/pattern-finding skills. One type of word puzzle that is particularly mindless, but for that very reason I still enjoy in the above circumstances, is a word search; you are given a grid with letters and/or numbers, and a list of “hidden” terms, and you circle them within the grid, crossing them off the list as you go along. I do admit that the categories of terms used in the typical word searches can become rather mundane (breeds of dog, types of food, words that are followed by “stone,” words associated with a famous movie star, words from a particular television show, etc.). Therefore, on one of my last seminar trips I decided to generate my own word search, using the category of autophagy.     

The intense gravitational attraction of autophagy

In the course of my work as Autophagy editor, I try to gauge the overall patterns of interest in autophagy research. Not surprisingly, the number of papers associated with this topic has increased steadily. However, that trend provides only one glimpse into the way interest in this field has been changing—that the number of people working on autophagy has expanded. Perhaps not surprisingly, the number of different research areas that now include autophagy studies is also increasing. Thus, I decided to carry out an informal, imprecise analysis of the number of different journals (presumably reflecting in part the number of topics) that include papers on autophagy.     

Activation of autophagy is required for muscle homeostasis during physical exercise

Skeletal muscle fibers of collagen VI null (Col6a1?/?) mice show signs of degeneration due to a block in autophagy, leading to the accumulation of damaged mitochondria and excessive apoptosis. Attempts to induce autophagic flux by subjecting these mutant mice to long-term or shorter bursts of physical activity are unsuccessful (see Grumati, et al., pp. 1415–23). In normal mice, the induction of autophagy in the skeletal muscles post-exercise is able to prevent the accumulation of damaged organelles and maintain cellular homeostasis. Thus, these studies provide an important connection between autophagy and exercise physiology.     

Activation of autophagy is required for muscle homeostasis during physical exercise

Skeletal muscle fibers of collagen VI null (Col6a12/2) mice show signs of degeneration due to a block in autophagy, leading to the accumulation of damaged mitochondria and excessive apoptosis. Attempts to induce autophagic flux by subjecting these mutant mice to long-term or shorter bursts of physical activity are unsuccessful (see Grumati, et al., pp. 1415–23). In normal mice, the induction of autophagy in the skeletal muscles post-exercise is able to prevent the accumulation of damaged organelles and maintain cellular homeostasis. Thus, these studies provide an important connection between autophagy and exercise physiology.     

Protocols,Toolboxes and Resource papers

In the August 2009 issue of Autophagy, I indicated that we were launching a new category of article, Protocols. At that time, I noted that we would ultimately be placing these articles on a new site online. Well, that time has finally arrived (see www.landesbioscience.com/journals/autophagy/protocols/ for links to these papers). Therefore, it seems appropriate for me to briefly distinguish among three types of community-oriented papers, Protocol, Toolbox and Resource.     

Downregulation of autophagy through CUL3-KLHL20-mediated turnover of the ULK1 and PIK3C3/VPS34 complexes

The molecular mechanism of macroautophagy/autophagy induction has been intensively studied, but little is known about downregulation of autophagy and how this process is restricted. In particular, how is autophagy maintained at an appropriate homeostatic level when cells are subjected to prolonged stress? In this study (see the related punctum in Autophagy 12–5), Liu et al. report a function of the CUL3-KLHL20 ubiquitin ligase in feedback regulation, leading to the downregulation of autophagy through the degradation of the ULK1 and PIK3C3/VPS34 complexes.     

Protocols, Toolboxes and Resource papers

In the August 2009 issue of Autophagy, I indicated that we were launching a new category of article, Protocols. At that time, I noted that we would ultimately be placing these articles on a new site online. Well, that time has finally arrived (see www.landesbioscience.com/journals/autophagy/protocols/ for links to these papers). Therefore, it seems appropriate for me to briefly distinguish among three types of community-oriented papers, Protocol, Toolbox and Resource.     

Approaching the molecular mechanism of autophagy

Autophagy is a complex cellular process that involves dynamic membrane rearrangements under a range of physiological conditions. It is a highly regulated process that plays a role in cellular maintenance and development, and has been implicated in a number of genetic diseases. Upon induction of autophagy, cytoplasm is sequestered into vesicles and delivered to a degradative organelle, the vacuole in yeast or the lysosome in mammalian cells. The process is unique in that it converts material that is topologically intracellular into topologically extracellular. Autophagy was first described more than 50 years ago, but it is since the discovery of the pathway in yeast cells that our knowledge about the molecular events taking place during the process has expanded. The generation of autophagy-specific mutants in a variety of yeast cell lines has provided insight into functional roles of more than 15 novel genes, double that number if we include genes whose products function also in other processes. Although we have learned much about autophagy, many questions remain to be answered. This review highlights the most recent advances in the autophagy field in both yeast and mammalian cells.     

Guidelines for the use and interpretation of assays for monitoring autophagy

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. 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 vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased 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. 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. 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 autophagy assays, we hope to encourage technical innovation in the field.     

Guidelines for the use and interpretation of assays for monitoring autophagy

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. 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 vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased 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. 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. 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 autophagy assays, we hope to encourage technical innovation in the field.     

The Atg17-Atg31-Atg29 complex and Atg11 regulate autophagosome-vacuole fusion

The macroautophagy (hereafter autophagy) process involves de novo formation of double-membrane autophagosomes; after sequestering cytoplasm these transient organelles fuse with the vacuole/lysosome. Genetic studies in yeasts have characterized more than 40 autophagy-related (Atg) proteins required for autophagy, and the majority of these proteins play roles in autophagosome formation. The fusion of autophagosomes with the vacuole is mediated by the Rab GTPase Ypt7, its guanine nucleotide exchange factor Mon1-Ccz1, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. However, these factors are not autophagosome-vacuole fusion specific. We recently showed that 2 autophagy scaffold proteins, the Atg17-Atg31-Atg29 complex and Atg11, regulate autophagosome-vacuole fusion by recruiting the vacuolar SNARE Vam7 to the phagophore assembly site (PAS), where an autophagosome forms in yeast.     

The vacuole vs. the lysosome

The morphometric examination of autophagic bodies provides useful information about the mechanism and magnitude of macroautophagy, and yeast researchers frequently utilize various measurements of these structures as part of their quantification of the process. The utility of this approach, however, has led to the common misconception that autophagic bodies can be found in the mammalian lysosome, which is generally not correct.     

The proteasome subunit RPN10 functions as a specific receptor for degradation of the 26S proteasome by macroautophagy in Arabidopsis

The ubiquitin-proteasome system (UPS) and macroautophagy/autophagy are 2 main degradative routes, which are important for cellular homeostasis. In a study conducted by Marshall et al., the authors demonstrated that the UPS and autophagy converge in Arabidopsis (see the punctum in issue #11–10). In particular, they found that the 26S proteasome is degraded by autophagy, either nonselectively (induced by nitrogen starvation) or selectively (induced by proteasome inhibition). The selective phenotype is mediated through the proteasome subunit RPN10, which can bind both ubiquitin and ATG8. This newly identified autophagic degradation of the proteasome is termed “proteaphagy,” and the process reveals an interesting relationship between these degradative systems.     

Editorial: The vacuole vs. the lysosome: When size matters

The morphometric examination of autophagic bodies provides useful information about the mechanism and magnitude of macroautophagy, and yeast researchers frequently utilize various measurements of these structures as part of their quantification of the process. The utility of this approach, however, has led to the common misconception that autophagic bodies can be found in the mammalian lysosome, which is generally not correct.