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

Autophagy plays an important role in immunity to microbial pathogens. The autophagy system can target bacteria in phagosomes, promoting phagosome maturation and preventing pathogen escape into the cytosol. Recently, Toll-like receptor (TLR) signaling from phagosomes was found to initiate their targeting by the autophagy system, but the mechanism by which TLR signaling activates autophagy is unclear. Here we show that autophagy targeting of phagosomes is not exclusive to those containing TLR ligands. Engagement of either TLRs or the Fc␥ receptors (Fc␥Rs) during phagocytosis induced recruitment of the autophagy protein LC3 to phagosomes with similar kinetics. Both receptors are known to activate the NOX2 NADPH oxidase, which plays a central role in microbial killing by phagocytes through the generation of reactive oxygen species (ROS). We found that NOX2-generated ROS are necessary for LC3 recruitment to phagosomes. Antibacterial autophagy in human epithelial cells, which do not express NOX2, was also dependent on ROS generation. These data reveal a coupling of oxidative and nonoxidative killing activities of the NOX2 NADPH oxidase in phagocytes through autophagy. Furthermore, our results suggest a general role for members of the NOX family in regulating autophagy.phagosome ͉ reactive oxygen species ͉ TLR ͉ innate immunity ͉ Salmonella

Autophagy is a cellular process that targets proteins, lipids and organelles to lysosomes for degradation, but it has also been shown to combat infection with various pathogenic bacteria. In turn, bacteria have developed diverse strategies to avoid autophagy by interfering with autophagy signalling or the autophagy machinery and, in some cases, they even exploit autophagy for their growth. In this Review, we discuss canonical and non-canonical autophagy pathways and our current knowledge of antibacterial autophagy, with a focus on the interplay between bacterial factors and autophagy components.

Macroautophagy (hereafter autophagy) is a ubiquitous process in eukaryotic cells that is integrally involved in various aspects of cellular and organismal physiology. The morphological hallmark of autophagy is the formation of double-membrane cytosolic vesicles, autophagosomes, which sequester cytoplasmic cargo and deliver it to the lysosome or vacuole. Thus, autophagy involves dynamic membrane mobilization, yet the source of the lipid that forms the autophagosomes and the mechanism of membrane delivery are poorly characterized. The TRAPP complexes are multimeric guanine nucleotide exchange factors (GEFs) that activate the Rab GTPase Ypt1, which is required for secretion. Here we describe another form of this complex (TRAPPIII) that acts as an autophagy-specific GEF for Ypt1. The Trs85 subunit of the TRAPPIII complex directs this Ypt1 GEF to the phagophore assembly site (PAS) that is involved in autophagosome formation. Consistent with the observation that a Ypt1 GEF is directed to the PAS, we find that Ypt1 is essential for autophagy. This is an example of a Rab GEF that is specifically targeted for canonical autophagosome formation.utophagy is a catabolic process in which damaged or superfluous cytoplasmic components are degraded in response to stress conditions; it is evolutionarily conserved in eukaryotes and is integrally involved in development and physiology (1, 2). The morphological hallmark of autophagy is the formation of doublemembrane cytosolic vesicles, autophagosomes, which sequester cytoplasm. The autophagosomes then fuse with the lysosome, resulting in the degradation of the cargo. The mechanism of autophagosome formation is distinct from that used for vesicle formation in the secretory or endocytic pathways and is said to be de novo in that it does not occur by direct budding from a preexisting organelle. Instead, a nucleating structure, the phagophore, appears to expand by the addition of membrane possibly through vesicular fusion. One consequence of this mechanism is that it allows the sequestration of essentially any sized cargo, including intact organelles or invasive microbes, and this capability is critical to autophagic function. When autophagy is induced there is a substantial demand for membrane, and a major question in the field concerns the membrane origin; nearly every organelle has been implicated in this role (3). The early secretory pathway is likely one such membrane source for autophagy (4, 5).Rab GTPases are key regulators of membrane traffic that mediate multiple events including vesicle tethering and membrane fusion. These molecular switches cycle between an inactive (GDP-bound) and active (GTP-bound) conformation. The yeast Rab Ypt1, which is essential for ER-Golgi and Golgi traffic (6), is activated by the multimeric guanine nucleotide exchange factor (GEF) called TRAPP (7,8). Two forms of the TRAPP complexes have been identified (9). These two complexes share several subunits, including four (Bet3, Bet5, Trs23, and Trs31) that are essential to activate Ypt1. How each of th...

The conventional inorganic semiconductors are not suitable for in vivo therapeutic nanomedicine because of the lack of an adequate and safe irradiation source to activate them. This work reports on the rational design of titania (TiO)-based semiconductors for enhanced and synergistic sono-/photoinduced tumor eradication by creating an oxygen-deficient TiO layer onto the surface of TiO nanocrystals, which can create a crystalline-disordered core/shell structure (TiO@TiO) with black color. As found in the lessons from traditional photocatalysis, such an oxygen-deficient TiO layer with abundant oxygen defects facilitates and enhances the separation of electrons (e) and holes (h) from the energy-band structure upon external ultrasound irradiation, which can significantly improve the efficacy of sono-triggered sonocatalytic tumor therapy. Such an oxygen-deficient TiO layer can also endow black titania nanoparticles with high photothermal-conversion efficiency (39.8%) at the NIR-II biowindow (1064 nm) for enhanced photothermal hyperthermia. Both in vitro cell level and systematic in vivo tumor-bearing mice xenograft evaluations have demonstrated the high synergistic efficacy of combined and enhanced sonodynamic therapy and photothermal ablation as assisted by oxygen-deficient black titania, which has achieved complete tumor eradication with high therapeutic biosafety and without obvious reoccurrence. This work not only provides the paradigm of high therapeutic efficacy of a combined sono-/photoinduced tumor-treatment protocol but also significantly broadens the nanomedical applications of semiconductor-based nanoplatforms by rational design of their nanostructures and control of their physiochemical properties.

Two-dimensional (2D) MXenes, as a new 2D functional material nanosystem, have been extensively explored for broad applications. However, their specific performance and applications in theranostic nanomedicine have still rarely been explored. This work reports on the drug-delivery performance and synergistic therapeutic efficiency of Ti C MXenes for highly efficient tumor eradication. These Ti C MXenes not only possess high drug-loading capability of as high as 211.8%, but also exhibit both pH-responsive and near infrared laser-triggered on-demand drug release. Especially, based on the high photothermal-conversion capability of Ti C MXenes, they have been further explored for efficient tumor eradication by synergistic photothermal ablation and chemotherapy, which has been systematically demonstrated both in vitro and in vivo. These Ti C MXenes have also been demonstrated as the desirable contrast agents for photoacoustic imaging, showing the potential for diagnostic-imaging guidance and monitoring during therapy. The high in vivo histocompatibility of Ti C and their easy excretion out of the body have been evaluated and demonstrated, showing the potential high biosafety for further clinical translation. This work paves a new way for broadening biomedical applications of MXenes in nanomedicine where they can exert the high performance and functionality for synergistic therapy, especially on combating cancer.

As a conserved cellular degradative pathway in eukaryotes, autophagy relieves cells from various types of stress. There are different forms of autophagy, and the ongoing studies of the molecular mechanisms and cellular functions of these processes are unraveling their significant roles in human health. Currently, the best-studied of these pathways is macroautophagy, which is linked to a range of human disease. For example, as part of the host immune defense mechanism, macroautophagy is activated to eliminate invasive pathogenic bacteria; however, in some cases bacteria subvert this process for their own replication. Autophagy also contributes to endogenous major histocompatibility complex class II antigen presentation, reflecting its role in adaptive immunity. In certain neurodegenerative diseases, which are associated with aggregation-prone proteins, macroautophagy plays a protective role in preventing or reducing cytotoxicity by clearance of the toxic proteins; however, the autophagy-dependent processing of some components correlates with the pathogenesis of certain myopathies. Finally, autophagy acts as a mechanism for tumor suppression, although some cancer cells use it as a cytoprotective mechanism. Thus, a fundamental paradox of autophagy is that it can act to promote both cell survival and cell death, depending on the specific conditions.

In response to stress conditions (such as nutrient limitation or accumulation of damaged organelles) and certain pathological situations, eukaryotic cells use autophagy as a survival mechanism. During nutrient stress the main purpose of autophagy is to degrade cytoplasmic materials within the lysosome/vacuole lumen and generate an internal nutrient pool that is recycled back to the cytosol. This study elucidates a molecular mechanism for linking the degradative and recycling roles of autophagy. We show that in contrast to published studies, Atg22 is not directly required for the breakdown of autophagic bodies within the lysosome/vacuole. Instead, we demonstrate that Atg22, Avt3, and Avt4 are partially redundant vacuolar effluxers, which mediate the efflux of leucine and other amino acids resulting from autophagic degradation. The release of autophagic amino acids allows the maintenance of protein synthesis and viability during nitrogen starvation. We propose a "recycling" model that includes the efflux of macromolecules from the lysosome/vacuole as the final step of autophagy.