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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Porphyromonas gingivalis is a periodontal pathogen that also localizes to atherosclerotic plaques. Our previous studies demonstrated that P. gingivalis is capable of invading endothelial cells and that intracellular bacteria are contained in vacuoles that resemble autophagosomes. In this study, we have examined the trafficking of P. gingivalis 381 to the autophagic pathway. P. gingivalis 381 internalized by human coronary artery endothelial (HCAE) cells is located within vacuoles morphologically identical to autophagosomes. The progression of P. gingivalis 381 through intracellular vacuoles was analyzed by immunofluorescence microscopy. Vacuoles containing P. gingivalis colocalize with Rab5 and HsGsa7p early after internalization. At later times, P. gingivalis colocalizes with BiP and then progresses to a vacuole that contains BiP and lysosomal glycoprotein 120. Late endosomal markers and the lysosomal cathepsin L do not colocalize with P. gingivalis 381. The intracellular survival of P. gingivalis 381 decreases over 8 h in HCAE cells pretreated with the autophagy inhibitors 3-methyladenine and wortmannin. In addition, the vacuole containing P. gingivalis 381 lacks BiP but contains cathepsin L in the presence of wortmannin. These results suggest that P. gingivalis 381 evades the endocytic pathway to lysosomes and instead traffics to the autophagosome.Porphyromonas gingivalis is a gram-negative, anaerobic rod that is considered to be among the major pathogens associated with adult periodontitis (64). A possible mechanism of pathogenesis may be cellular invasion. P. gingivalis has been demonstrated to be internalized within gingival epithelial cells in vitro (19,34,53) and buccal epithelial cells in vivo (51). Recent epidemiological studies have demonstrated a strong relationship between periodontal disease and coronary heart disease (2,3,17,38,39). Oral bacteria have a direct route to the circulatory system in periodontitis patients due to transient bacteremias produced by flossing, mastication, and toothbrushing (11,57,62). P. gingivalis localizes to atherosclerotic plaques (12,30) and is capable of invasion of coronary artery cells in vitro (16,18). Therefore, invasion and intracellular parasitism of endothelial cells by P. gingivalis in vivo may exacerbate the inflammatory response of atherosclerosis.Invasion of nonphagocytic cells is a common strategy of evading the immune system for many pathogens (23). Once within the cell, these pathogens have developed various mechanisms for survival (28,40). Legionella pneumophila and virulent Brucella abortus gain access to and replicate in vacuoles that resemble autophagosomes and are associated with endoplasmic reticulum proteins (46, 47). Autophagosomes, multimembranous vacuoles formed from invaginations of ribosome-free regions of the rough endoplasmic reticulum (RER) (20), are the organelles of the autophagic process. Autophagy is a process whereby cytosol and organelles are sequestered for lysosome degradation in response to nutrient deprivation (20). Under normal...