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

Beclin 1, the mammalian orthologue of yeast Atg6, has a central role in autophagy, a process of programmed cell survival, which is increased during periods of cell stress and extinguished during the cell cycle. It interacts with several cofactors (Atg14L, UVRAG, Bif-1, Rubicon, Ambra1, HMGB1, nPIST, VMP1, SLAM, IP 3 R, PINK and survivin) to regulate the lipid kinase Vps-34 protein and promote formation of Beclin 1-Vps34-Vps15 core complexes, thereby inducing autophagy. In contrast, the BH3 domain of Beclin 1 is bound to, and inhibited by Bcl-2 or Bcl-XL. This interaction can be disrupted by phosphorylation of Bcl-2 and Beclin 1, or ubiquitination of Beclin 1. Interestingly, caspase-mediated cleavage of Beclin 1 promotes crosstalk between apoptosis and autophagy. Beclin 1 dysfunction has been implicated in many disorders, including cancer and neurodegeneration. Here, we summarize new findings regarding the organization and function of the Beclin 1 network in cellular homeostasis, focusing on the cross-regulation between apoptosis and autophagy. Cell Death and Differentiation (2011) 18, 571-580; doi:10.1038/cdd.2010 published online 11 February 2011 Autophagy is an essential process that consists of selective degradation of cellular components. There are at least three different types of autophagy described and possibly more. These autophagy types include macroautophagy (hereafter referred to as autophagy), microautophagy and chaperonemediated autophagy. 1 The initial step of autophagy is the surrounding and sequestering of cytoplasmic organelles and proteins within an isolation membrane (phagophore). Potential sources for the membrane to generate the phagophore include the Golgi complex, endosomes, the endoplasmic reticulum (ER), mitochondria and the plasma membrane. 2 The nascent membranes are fused at their edges to form double-membrane vesicles, called autophagosomes. Autophagosomes undergo a stepwise maturation process, including fusion with acidified endosomal and/or lysosomal vesicles, eventually leading to the delivery of cytoplasmic contents to lysosomal components, where they fuse, then degrade and are recycled (Figure 1a). The process of mammalian autophagy is divided into six principal steps: initiation (Figure 1b It has been well demonstrated that autophagy depends on Atg5/Atg7, is associated with microtubule-associated protein light chain 3 (LC3) truncation and lipidation, and may originate directly from the ER membrane and other membrane organelles. Furthermore, recent study has identified a Atg5/Atg7-independent pathway of autophagy. 3 This pathway of autophagy was not associated with LC3 processing but appeared to involve autophagosome formation from late endosomes and the trans-Golgi. 3 Atg7-independent autophagy had been implicated in mitochondrial clearance from reticulocytes. 4 The exact molecular basis of Atg5/Atg7-independent autophagy remains to be elucidated. Interestingly, Beclin 1 is required for Atg5/Atg7-dependent and -independent autophagy. 3 However, the presence of Beclin 1-indep...

Macroautophagy/autophagy is an evolutionarily conserved degradation pathway that maintains homeostasis. Ferroptosis, a novel form of regulated cell death, is characterized by a production of reactive oxygen species from accumulated iron and lipid peroxidation. However, the relationship between autophagy and ferroptosis at the genetic level remains unclear. Here, we demonstrated that autophagy contributes to ferroptosis by degradation of ferritin in fibroblasts and cancer cells. Knockout or knockdown of Atg5 (autophagy-related 5) and Atg7 limited erastin-induced ferroptosis with decreased intracellular ferrous iron levels, and lipid peroxidation. Remarkably, NCOA4 (nuclear receptor coactivator 4) was a selective cargo receptor for the selective autophagic turnover of ferritin (namely ferritinophagy) in ferroptosis. Consistently, genetic inhibition of NCOA4 inhibited ferritin degradation and suppressed ferroptosis. In contrast, overexpression of NCOA4 increased ferritin degradation and promoted ferroptosis. These findings provide novel insight into the interplay between autophagy and regulated cell death.

Complex genetic and physiological variations as well as environmental factors that drive emergence of chromosomal instability, development of unscheduled cell death, skewed differentiation, and altered metabolism are central to the pathogenesis of human diseases and disorders. Understanding the molecular bases for these processes is important for the development of new diagnostic biomarkers, and for identifying new therapeutic targets. In 1973, a group of non-histone nuclear proteins with high electrophoretic mobility was discovered and termed High-Mobility Group (HMG) proteins. The HMG proteins include three superfamilies termed HMGB, HMGN, and HMGA. High-mobility group box 1 (HMGB1), the most abundant and well-studied HMG protein, senses and coordinates the cellular stress response and plays a critical role not only inside of the cell as a DNA chaperone, chromosome guardian, autophagy sustainer, and protector from apoptotic cell death, but also outside the cell as the prototypic damage associated molecular pattern molecule (DAMP). This DAMP, in conjunction with other factors, thus has cytokine, chemokine, and growth factor activity, orchestrating the inflammatory and immune response. All of these characteristics make HMGB1 a critical molecular target in multiple human diseases including infectious diseases, ischemia, immune disorders, neurodegenerative diseases, metabolic disorders, and cancer. Indeed, a number of emergent strategies have been used to inhibit HMGB1 expression, release, and activity in vitro and in vivo. These include antibodies, peptide inhbitiors, RNAi, anti-coagulants, endogenous hormones, various chemical compounds, HMGB1-receptor and signaling pathway inhibition, artificial DNAs, physical strategies including vagus nerve stimulation and other surgical approaches. Future work further investigating the details of HMGB1 localizationtion, structure, post-translational modification, and identifccation of additional partners will undoubtedly uncover additional secrets regarding HMGB1’s multiple functions.

Summary Pathogen-associated molecular pattern molecules (PAMPs) are derived from microorganisms and recognized by pattern recognition receptor (PRR)-bearing cells of the innate immune system as well as many epithelial cells. In contrast, damage-associated molecular pattern molecules (DAMPs) are cell-derived and initiate and perpetuate immunity in response to trauma, ischemia, and tissue damage, either in the absence or presence of pathogenic infection. Most PAMPs and DAMPs serve as so-called ‘Signal 0s’ that bind specific receptors [Toll-like receptors, NOD-like receptors, RIG-I-like receptors, AIM2-like receptors, and the receptor for advanced glycation end products (RAGE)] to promote autophagy. Autophagy, a conserved lysosomal degradation pathway, is a cell survival mechanism invoked in response to environmental and cellular stress. Autophagy is inferred to have been present in the last common eukaryotic ancestor and only to have been lost by some obligatory intracellular parasites. As such, autophagy represents a unifying biology, subserving survival and the earliest host defense strategies, predating apoptosis, within eukaryotes. Here, we review recent advances in our understanding of autophagic molecular mechanisms and functions in emergent immunity.