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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.

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.

We investigated the role of Atg1 in autophagic cell death (ACD) in a Dictyostelium monolayer model. The model is especially propitious, not only because of genetic tractability and absence of apoptosis machinery, but also because induction of ACD requires two successive exogenous signals, first the combination of starvation and cAMP, second the differentiation factor DIF-1. This enables one to analyze separately first-signal-induced autophagy and subsequent second-signal-induced ACD. We used mutants of atg1, a gene that plays an essential role in the initiation of autophagy. Upon starvation/cAMP, in contrast to parental cells, atg1 mutant cells showed irreversible lesions, clearly establishing a protective role for Atg1. Upon subsequent exposure to DIF-1 or to more ACD-specific second signals, starved parental cells progressed to ACD, but starved atg1 mutant cells did not, showing that Atg1 was required for ACD. Thus, in the same cells Atg1 was required in two apparently opposite ways, upon first-signaling for cell survival and upon second-signaling for ACD. Our findings strongly suggest that Atg1, thus presumably autophagy, protects the cells from starvation-induced cell death, allowing subsequent induction of ACD by the second signal. ACD is therefore not only "with" autophagy (since it showed signs of autophagy throughout), but is also "allowed by" autophagy. This does not exclude a role for autophagy also after second signaling. These results may account for discrepancies reported in the literature, encourage searches for second signals in different developmental models of ACD, and incite caution in autophagy-related therapeutic attempts.

The use of simple organisms to understand the molecular and cellular function of complex processes is instrumental for the rapid development of biomedical research. A remarkable example has been the discovery in S. cerevisiae of a group of proteins involved in the pathways of autophagy. Orthologues of these proteins have been identified in humans and experimental model organisms. Interestingly, some mammalian autophagy proteins do not seem to have homologues in yeast but are present in Dictyostelium, a social amoeba with two distinctive life phases, a unicellular stage in nutrient-rich conditions that differentiates upon starvation into a multicellular stage that depends on autophagy. This review focuses on the identification and annotation of the putative Dictyostelium autophagy genes and on the role of autophagy in development, cell death and infection by bacterial pathogens.

Dictyostelium cells in monolayers in vitro lend themselves well to a study of autophagic cell death (ACD). There is no apoptosis machinery in the protist Dictyostelium, no caspase nor Bcl-2 family members (except a paracaspase whose inactivation does not alter cell death), thus there is no apoptosis that could interfere with, or substitute for, nonapoptotic cell death. Also, Dictyostelium, a eukaryote, has a haploid genome, which facilitates random insertional mutagenesis.

Dictyostelium constitutes a genetically tractable model for the analysis of autophagic cell death (ACD). During ACD, Dictyostelium cells first transform into paddle cells and then become round, synthesize cellulose, vacuolize, and die. Through random insertional mutagenesis, we identified the receptor histidine kinase DhkM as being essential for ACD. Surprisingly, different DhkM mutants showed distinct nonvacuolizing ACD phenotypes. One class of mutants arrested ACD at the paddle cell stage, perhaps through a dominant-negative effect. Other mutants, however, progressed further in the ACD program. They underwent rounding and cellulose synthesis but stopped before vacuolization. Moreover, they underwent clonogenic but not morphological cell death. Exogenous 8-bromo-cAMP restored vacuolization and death. A role for a membrane receptor at a late stage of the ACD pathway is puzzling, raising questions as to which ligand it is a receptor for and which moieties it phosphorylates. Together, DhkM is the most downstream-known molecule required for this model ACD, and its distinct mutants genetically separate previously undissociated late cell death events.

Autophagic cell death (ACD) can be operationally described as cell death with an autophagic component. While most molecular bases of this autophagic component are known, in ACD the mechanism of cell death proper is not well defined, in particular because in animal cells there is poor experimental distinction between what triggers autophagy and what triggers ACD. Perhaps as a consequence, it is often thought that in animal cells a little autophagy is protective while a lot is destructive and leads to ACD, thus that the shift from autophagy to ACD is quantitative. The aim of this article is to review current knowledge on ACD in Dictyostelium, a very favorable model, with emphasis on (1) the qualitative, not quantitative nature of the shift from autophagy to ACD, in contrast to the above, and (2) random or targeted mutations of in particular the following genes: iplA (IP3R), TalB (talinB), DcsA (cellulose synthase), GbfA, ugpB, glcS (glycogen synthase) and atg1. These mutations allowed the genetic dissection of ACD features, dissociating in particular vacuolisation from cell death.

Autophagic or necrotic cell death (ACD and NCD, respectively), studied in the model organism Dictyostelium which offers unique advantages, require triggering by the same differentiation-inducing factor DIF-1. To initiate these two types of cell death, does DIF-1 act through only one or through two distinct recognition structures? Such distinct structures may recognize distinct motifs of DIF-1. To test this albeit indirectly, DIF-1 was modified at one or two of several positions, and the corresponding derivatives were tested for their abilities to induce ACD or NCD. The results strongly indicated that distinct biochemical motifs of DIF-1 were required to trigger ACD or NCD, and that these motifs were separately recognized at the onset of ACD or NCD. In addition, both ACD and NCD were induced more efficiently by DIF-1 than by either its precursors or its immediate catabolite. These results showed an unexpected relation between a differentiation factor, the cellular structures that recognize it, the cell death types it can trigger and the metabolic state of the cell. The latter seems to guide the choice of the signaling pathway to cell death, which in turn imposes the cell death type and the recognition pattern of the differentiation factor.

Autophagic cell death in Dictyostelium can be dissociated into a starvation-induced sensitization stage and a death induction stage. A UDP-glucose pyrophosphorylase (ugpB) mutant and a glycogen synthase (glcS) mutant shared the same abnormal phenotype. In vitro, upon starvation alone mutant cells showed altered contorted morphology, indicating that the mutations affected the pre-death sensitization stage. Upon induction of cell death, most of these mutant cells underwent death without vacuolization, distinct from either autophagic or necrotic cell death. Autophagy itself was not grossly altered as shown by conventional and electron microscopy. Exogenous glycogen or maltose could complement both ugpB(-) and glcS(-) mutations, leading back to autophagic cell death. The glcS(-) mutation could also be complemented by 2-deoxyglucose that cannot undergo glycolysis. In agreement with the in vitro data, upon development glcS(-) stalk cells died but most were not vacuolated. We conclude that a UDP-glucose derivative (such as glycogen or maltose) plays an essential energy-independent role in autophagic cell death.

The signaling pathways governing pathophysiologically important autophagic (ACD) and necrotic (NCD) cell death are not entirely known. In the Dictyostelium eukaryote model, which benefits from both unique analytical and genetic advantages and absence of potentially interfering apoptotic machinery, the differentiation factor DIF leads from starvation-induced autophagy to ACD, or, if atg1 is inactivated, to NCD. Here, through random insertional mutagenesis, we found that inactivation of the iplA gene, the only gene encoding an inositol 1,4,5-trisphosphate receptor (IP3R) in this organism, prevented ACD. The IP3R is a ligand-gated channel governing Ca(2+) efflux from endoplasmic reticulum stores to the cytosol. Accordingly, Ca(2+)-related drugs also affected DIF signaling leading to ACD. Thus, in this system, a main pathway signaling ACD requires IP3R and further Ca(2+)-dependent steps. This is one of the first insights in the molecular understanding of a signaling pathway leading to autophagic cell death.

Autophagic cell death can be conveniently studied in Dictyostelium discoideum, an exceptionally favorable model not only because of its well-known genetic and experimental advantages but also because in Dictyostelium there is no apoptosis machinery that could interfere with nonapoptotic cell death. Moreover, autophagic cell death in Dictyostelium can be dissociated into a starvation-induced sensitization stage, during which autophagy is induced, and a death induction stage. We show here how to demonstrate, assess and analyze this autophagic cell death. This can be studied in vivo during the development of Dictyostelium, and in vitro, using modifications of the monolayer technique of Rob Kay et al. Methods to follow this autophagic cell death qualitatively and quantitatively are reported.

Non-apoptotic cell death types can be conveniently studied in Dictyostelium discoideum, an exceptionally favorable model not only because of its well-known genetic and experimental advantages, but also because in Dictyostelium there is no apoptosis machinery that could interfere with non-apoptotic cell death. We show here how to conveniently demonstrate, assess, and study these non-apoptotic cell death types. These can be generated by use of modifications of the monolayer technique of Rob Kay et al., and either wild-type HMX44A Dictyostelium cells, leading to autophagic cell death, or the corresponding atg1- autophagy gene mutant cells, leading to necrotic cell death. Methods to follow these non-apoptotic cell death types qualitatively and quantitatively will be reported.

Among unusual models to study cell death mechanisms, the protist Dictyostelium is remarkable because of its strategic phylogenetic position, with early emergence among eukaryotes and unicellular/multicellular transition, and its very favorable experimental and genetic flexibility. Dictyostelium shows developmental vacuolar cell death, and in vitro monolayer approaches revealed both an autophagic vacuolar and a necrotic type of cell death. These are described in some detail, as well as implications and future prospects.

In this chapter, we describe how to conveniently demonstrate, assess, and study cell death in Dictyostelium through simple cell culture, clonogenic tests, and photonic (with the help of staining techniques) and electronic microscopy. Cell death can be convniently generated using minor modifications of the monolayer technique of Rob Kay et al., and either wild-type HMX44A Dictyostelium cells or the corresponding atg1- autophagy gene mutant cells. Methods to follow cell death qualitatively and quantitatively facilitate detailed studies of vacuolar death in wild-type cells and of nonvacuolar, "condensed" death in atg1- mutant cells.

Types of cell death include apoptosis, necrosis, and autophagic cell death. The latter can be defined as death of cells containing autophagosomes, autophagic bodies, and/or vacuoles. Are autophagy and vacuolization causes, consequences, or side effects in cell death with autophagy? Would control of autophagy suffice to control this type of cell death? We disrupted the atg1 autophagy gene in Dictyostelium discoideum, a genetically tractable model for developmental autophagic vacuolar cell death. The procedure that induced autophagy, vacuolization, and death in wild-type cells led in atg1 mutant cells to impaired autophagy and to no vacuolization, demonstrating that atg1 is required for vacuolization. Unexpectedly, however, cell death still took place, with a non-vacuolar and centrally condensed morphology. Thus, a cell death mechanism that does not require vacuolization can operate in this cell death model showing conspicuous vacuolization. The revelation of non-vacuolar cell death in this protist by autophagy gene disruption is reminiscent of caspase inhibition revealing necrotic cell death in animal cells. Thus, hidden alternative cell death pathways may be found across kingdoms and for diverse types of cell death.

Different types of cell death are often defined by morphological criteria, without a clear reference to precise biochemical mechanisms. The Nomenclature Committee on Cell Death (NCCD) proposes unified criteria for the definition of cell death and of its different morphologies, while formulating several caveats against the misuse of words and concepts that slow down progress in the area of cell death research. Authors, reviewers and editors of scientific periodicals are invited to abandon expressions like ‘percentage apoptosis’ and to replace them with more accurate descriptions of the biochemical and cellular parameters that are actually measured. Moreover, at the present stage, it should be accepted that caspase-independent mechanisms can cooperate with (or substitute for) caspases in the execution of lethal signaling pathways and that ‘autophagic cell death’ is a type of cell death occurring together with (but not necessarily by) autophagic vacuolization. This study details the 2009 recommendations of the NCCD on the use of cell death-related terminology including ‘entosis’, ‘mitotic catastrophe’, ‘necrosis’, ‘necroptosis’ and ‘pyroptosis’.Cell Death and Differentiation (2009) 16, 3–11; doi:10.1038/cdd.2008.150; published online 10 October 2008 [ABSTRACT FROM AUTHOR]