The two Dictyostelium autophagy eight proteins, ATG8a and ATG8b, associate with the autophagosome in succession
TL;DR: The data infer that Dictyostelium ATG8a and ATg8b have distinct functions in autophagosome formation and that ATG 8b is the functional orthologue of the mammalian LC3 subfamily and ATG7a of the GABARAP subfamily.
About: This article is published in European Journal of Cell Biology.The article was published on 2016-01-01 and is currently open access. It has received 16 citations till now. The article focuses on the topics: Autophagosome & ATG8.
Citations
More filters
01 Jan 2012
TL;DR: In this paper, the authors present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macro-autophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes.
Abstract: 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.
173 citations
TL;DR: In recent years the understanding of, and ability to investigate, autophagy in D. discoideum has evolved significantly and will surely enable and accelerate future research using this model.
Abstract: Autophagy is a fast-moving field with an enormous impact on human health and disease. Understanding the complexity of the mechanism and regulation of this process often benefits from the use of simple experimental models such as the social amoeba Dictyostelium discoideum. Since the publication of the first review describing the potential of D. discoideum in autophagy, significant advances have been made that demonstrate both the experimental advantages and interest in using this model. Since our previous review, research in D. discoideum has shed light on the mechanisms that regulate autophagosome formation and contributed significantly to the study of autophagy-related pathologies. Here, we review these advances, as well as the current techniques to monitor autophagy in D. discoideum. The comprehensive bioinformatics search of autophagic proteins that was a substantial part of the previous review has not been revisited here except for those aspects that challenged previous predictions such as the composition of the Atg1 complex. In recent years our understanding of, and ability to investigate, autophagy in D. discoideum has evolved significantly and will surely enable and accelerate future research using this model.
71 citations
Cites background from "The two Dictyostelium autophagy eig..."
...discoideum possess 2 Atg8 paralogs (Atg8a and b), which are both good markers of autophagosomes although they play partially nonredundant roles (discussed below).(32) Another useful marker is Atg18, the ortholog of mammalian WIPI2....
[...]
...discoideum cells expressing RFP-Atg8a and GFP-Atg8b showed that Atg8b associates with autophagosomes before ATG8a.(32) In mammals, LC3 subfamily members are involved in the elongation of the phagophore, whereas the GABARAP subfamily seem to be essential for a later stage in maturation....
[...]
...Live cell imaging of D. discoideum cells expressing RFP-Atg8a and GFP-Atg8b showed that Atg8b associates with autophagosomes before ATG8a.32 In mammals, LC3 subfamily members are involved in the elongation of the phagophore, whereas the GABARAP subfamily seem to be essential for a later stage in maturation.71,72 Thus, Atg8b is likely the functional ortholog of the mammalian LC3 subfamily, which is also supported by the grouping in the evolutionary tree (Fig....
[...]
...6), and Atg8a the ortholog of the GABARAP subfamily.(32) It appears therefore, that duplication of Atg8 early in eukaryotic evolution allowed specialization of...
[...]
...discoideum Atg8 have been generated.(32,35) While these antibodies are useful for many experiments, fixed samples lack dynamic information....
[...]
TL;DR: It is found that ATG5- and ATG7-mediated autophagy is essential for the differentiation and cytoplasmic reduction of the flagellated motile sperm and hence for sperm fertility, and the similarities between the need of macroautophagy for sperm differentiation in moss and mouse are striking.
Abstract: Autophagy, a major catabolic process in eukaryotes, was initially related to cell tolerance to nutrient depletion. In plants autophagy has also been widely related to tolerance to biotic and abioti ...
43 citations
Cites background from "The two Dictyostelium autophagy eig..."
...larger RFP-ATG8 structures were also detected in several Dictyostelium atg KO mutants and these were colocalized with ubiquitin, suggesting that they represent protein aggregates.(41,42) In the Arabidopsis atg4a atg4b double-mutant, globular GFP-ATG8 accumulations are soluble and were also suggested to be protein aggregates....
[...]
TL;DR: The social amoeba Dictyostelium discoideum is presented as a relevant model to study autophagy and several methods based on the tracking and observation of autophagosomes by microscopy, analysis of changes in expression of autophile genes and proteins, and examination of the autophagic flux with various techniques are discussed.
Abstract: Autophagy is a eukaryotic catabolic pathway that degrades and recycles cellular components to maintain homeostasis. It can target protein aggregates, superfluous biomolecular complexes, dysfunctional and damaged organelles, as well as pathogenic intracellular microbes. Autophagy is a dynamic process in which the different stages from initiation to final degradation of cargo are finely regulated. Therefore, the study of this process requires the use of a palette of techniques, which are continuously evolving and whose interpretation is not trivial. Here, we present the social amoeba Dictyostelium discoideum as a relevant model to study autophagy. Several methods have been developed based on the tracking and observation of autophagosomes by microscopy, analysis of changes in expression of autophagy genes and proteins, and examination of the autophagic flux with various techniques. In this review, we discuss the pros and cons of the currently available techniques to assess autophagy in this organism.
27 citations
Cites background from "The two Dictyostelium autophagy eig..."
...In addition, the steady-state protein levels of the two Dictyostelium Atg8 orthologues were recently analyzed by western blot, and were shown to have different expression patterns during development [48]....
[...]
...It is worth noting, however, that Dictyostelium possesses two Atg8 orthologues, which appear to play partly non-redundant roles in autophagosome formation, with different dynamics of recruitment [48,49]....
[...]
...It is worth notin , however, that D cty s elium possesse two Atg8 orthologues, which appear to play partly non-redundant roles in autophagosome formation, with differ nt dynamics f recruitment [48,49]....
[...]
...Additionally, as antibodies against the two Atg8 Dictyostelium orthologues have been generated, the endogenous proteins can be evaluated using immunohistochemistry [48,52]....
[...]
TL;DR: It is suggested that Cr(VI) can induce excessive autophagy in LMH cells, while PTA can ameliorate Cr( VI)-induced autophagic by inhibiting ER stress.
Abstract: This study aimed to investigate the role of purple tomato anthocyanin (PTA) in autophagy induced by chromium(VI) in a chicken hepatocellular carcinoma cell line (LMH cells). LMH cells were exposed to Cr(VI), PTA, and Cr(VI) + PTA. The changes in endoplasmic reticulum (ER) stress, autophagy, related proteins, and COX-2 were detected. Results showed that the cell viability was reduced after Cr(VI) treatment, and the decrease was also restrained by 3-MA or PTA. Levels of ER stress-related proteins (GRP78/Bip and PERK) and COX-2 increased after Cr(VI) treatment, which resulted in an increase in autophagy-related proteins (Beclin1 and LC3-II), inhibition of autophagy pathway protein mTOR, and degradation of autophagy-related protein p62, leading to excessive autophagy and cell damage. Meanwhile, the changes of these indicators induced by Cr(VI) were alleviated by PTA. In conclusion, our study suggested that Cr(VI) can induce excessive autophagy in LMH cells, while PTA can ameliorate Cr(VI)-induced autophagy by inhibiting ER stress.
23 citations
References
More filters
TL;DR: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products.
Abstract: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
232,912 citations
Journal Article•
TL;DR: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products as mentioned in this paper.
Abstract: Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
203,017 citations
TL;DR: A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets that results in quantitative transfer of ribosomal proteins from gels containing urea.
Abstract: A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.
53,030 citations
TL;DR: In this paper, the authors present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macro-autophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes.
Abstract: 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.
5,187 citations
Daniel J. Klionsky1, Fábio Camargo Abdalla2, Hagai Abeliovich3, Robert T. Abraham4 +1284 more•Institutions (463)
TL;DR: These guidelines are presented 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.
Abstract: 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.
4,316 citations