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

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SUMMARY Encystation and sporulation are crucial developmental transitions for solitary and social amoebas, respectively. While little is known of encystation, sporulation requires both extra- and intracellular cAMP. After aggregation of social amoebas, extracellular cAMP binding to surface receptors and intracellular cAMP binding to cAMP dependent protein kinase (PKA) act together to induce prespore differentiation. Later, a second episode of PKA activation triggers spore maturation. Adenylyl cyclase B (ACB) produces cAMP for maturation, but the cAMP source for prespore induction is unknown. We show that adenylyl cyclase G (ACG) protein is upregulated in prespore tissue after aggregation. acg null mutants show reduced prespore differentiation, which becomes very severe when ACB is also deleted. ACB is normally expressed in prestalk cells, but is upregulated in the prespore region of acg null structures. These data show that ACG induces prespore differentiation in wild-type cells, with ACB capable of partially taking over this function in its absence.

We report the characterization of an unusual adenylyl cyclase gene from Plasmodium falciparum, here designated PfAC␣. The level of mRNA expression is maximum during development of gametocytes (the sexual blood stage of the parasite life cycle). The gene is highly interrupted by 22 introns, and reverse transcriptase-PCR analysis revealed that there are multiple mRNA splice variants. One intron has three alternative 3-splice sites that confer the potential to encode distinct forms of the enzyme using alternative start codons. Deduced amino acid sequences predict membrane-spanning regions, the number of which can vary between two and six depending on the splice variant. Expression of a synthetic form of two of these variants in Xenopus oocytes and in Dictyostelium adenylyl cyclase-deficient mutants, confirms that PfAC␣ is a functional adenylyl cyclase. These results identify a novel mechanism in P. falciparum for the generation of multiple isoforms of a key, membranebound signaling molecule from a single genomic copy. Comparisons of the catalytic domains of PfAC␣ and a second putative P. falciparum adenylyl cyclase (PfAC␤) with those from other species reveal an unexpected similarity with adenylyl cyclases from certain prokaryotes including the cyanobacteria (blue green algae). In addition, the presence of an unusual active site substitution in a position that determines substrate specificity, also characteristic of these prokaryotic forms of the enzyme, further suggests a plastid origin for the Plasmodium cyclases.