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.

For example, 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 versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase 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. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

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. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. 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 assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Autophagy is a mechanism by which cellular material is delivered to lysosomes for degradation, leading to the basal turnover of cell components and providing energy and macromolecular precursors. Autophagy has opposing, context-dependent roles in cancer, and interventions to both stimulate and inhibit autophagy have been proposed as cancer therapies. This has led to the therapeutic targeting of autophagy in cancer to be sometimes viewed as controversial. In this Review, we suggest a way forwards for the effective targeting of autophagy by understanding the context-dependent roles of autophagy and by capitalizing on modern approaches to clinical trial design.

/pdf/targeting-autophagy-in-cancer-2pjcn0h04a.pdf

Targeting autophagy in cancer

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

Macroautophagy, initially described as a non-selective nutrient recycling process, is essential for the removal of multiple cellular components. In the past three decades, selective autophagy has been characterized as a highly regulated and specific degradation pathway for removal of unwanted cytosolic components and damaged and/or superfluous organelles. Here, we discuss different types of selective autophagy, emphasizing the role of ligand receptors and scaffold proteins in providing cargo specificity, and highlight unanswered questions in the field.

/pdf/cargo-recognition-and-degradation-by-selective-autophagy-256egoe5dv.pdf

Cargo recognition and degradation by selective autophagy

Autophagy is a conserved, self-degradation system that is critical for maintaining cellular homeostasis during stress conditions. Dysregulated autophagy has implications in health and disease. Specifically, in cancer, autophagy plays a dichotomous role by inhibiting tumor initiation but supporting tumor progression. Early results from clinical trials that repurposed hydroxychloroquine for cancer have suggested that autophagy inhibition may be a promising approach for advanced cancers. In this review of the literature, the authors present fundamental advances in the biology of autophagy, approaches to targeting autophagy, the preclinical rationale and clinical experience with hydroxychloroquine in cancer clinical trials, the potential role of autophagy in tumor immunity, and recent developments in next-generation autophagy inhibitors that have clinical potential. Autophagy is a promising target for drug development in cancer. Cancer 2018. © 2018 American Cancer Society.

https://acsjournals.onlinelibrary.wiley.com/doi/pdf/10.1002/cncr.31335

Autophagy is a primarily degradative pathway that takes place in all eukaryotic cells. It is used for recycling cytoplasm to generate macromolecular building blocks and energy under stress conditions, to remove superfluous and damaged organelles to adapt to changing nutrient conditions and to maintain cellular homeostasis. In addition, autophagy plays a critical role in cytoprotection by preventing the accumulation of toxic proteins and through its action in various aspects of immunity including the elimination of invasive microbes and its participation in antigen presentation. The most prevalent form of autophagy is macroautophagy, and during this process, the cell forms a double-membrane sequestering compartment termed the phagophore, which matures into an autophagosome. Following delivery to the vacuole or lysosome, the cargo is degraded and the resulting macromolecules are released back into the cytosol for reuse. The past two decades have resulted in a tremendous increase with regard to the molecular studies of autophagy being carried out in yeast and other eukaryotes. Part of the surge in interest in this topic is due to the connection of autophagy with a wide range of human pathophysiologies including cancer, myopathies, diabetes and neurodegenerative disease. However, there are still many aspects of autophagy that remain unclear, including the process of phagophore formation, the regulatory mechanisms that control its induction and the function of most of the autophagy-related proteins. In this review, we focus on macroautophagy, briefly describing the discovery of this process in mammalian cells, discussing the current views concerning the donor membrane that forms the phagophore, and characterizing the autophagy machinery including the available structural information.

/pdf/the-machinery-of-macroautophagy-3yn7cwcpxq.pdf

The machinery of macroautophagy

https://www.cell.com/current-biology/pdf/S0960-9822(14)00069-4.pdf

An Alternative Root for the Eukaryote Tree of Life

Program in Systematic Biology, Department of OrganismalBiology,UppsalaUniversity,Norbyva¨gen18D,75236Uppsala,SwedenSummaryTherootoftheeukaryotetreeoflifedeﬁnessomeofthemostfundamental relationships among species. It is also criticalfor deﬁning the last eukaryote common ancestor (LECA),the shared heritage of all extant species. The unikont-bikontroothasbeenthereigningparadigmforeukaryotesformorethan 10 years [1] but is becoming increasingly controversial[2–4].Wedevelopedacarefullyvetteddataset,consistingof37 nuclear-encoded proteins of close bacterial ancestry(euBacs) and their closest bacterial relatives, augmentedby deep sequencing of the Acrasis kona (Heterolobosea,Discoba) transcriptome. Phylogenetic analysis of thesedata produces a highly robust, fully resolved global phy-logenyofeukaryotes.Thetreesortsallexaminedeukaryotesinto three megagroups and identiﬁes the Discoba, andpotentially its parent taxon Excavata [5], as the sister groupto the bulk of known eukaryote diversity, the proposedNeozoa (Amorphea + Stramenopila+Alveolata+Rhizaria+Plantae [SARP] [6]). All major alternative hypotheses arerejected with as little as w50% of the data, and this resolu-tionisunaffectedbythepresenceoffast-evolvingalignmentpositions or distant outgroup sequences. This ‘‘neozoan-excavate’’ root revises hypotheses of early eukaryoteevolution and highlights the importance of the poorly stud-ied Discoba for understanding the evolution of eukaryoticdiversity and basic cellular processes.ResultsThirty-Seven Universal euBac ProteinsMultigene phylogenies now assign most known eukaryotesto a few major groups, and these to three proposedmegagroups—Amorphea, Stramenopila+Alveolata+Rhizaria+Plantae (SARP or Diaphoretickes), and Excavata [5]. Howev-er, little effort has been made to explore the eukaryote rootwith multigene phylogeny, and attempts to root the treewith macromolecular characters have given widely differentresults [1, 3, 4, 6]. We sought to develop a multigene dataset tailored to the study of deep eukaryote phylogeny,including a close outgroup to root the tree. Since mitochon-dria or their remnants are unique to eukaryotes and universalamong them [7], universal eukaryotic genes of bacterialorigin (euBacs) should provide one of the closest possibleoutgroups to root the tree.Two parallel protocols employing a combination of homolo-gous clustering and phylogenetic screening were used toidentify proteins suitable for deep eukaryote phylogeny (seeSupplemental Experimental Procedures and Figure S1available online). Screening identiﬁed genes that appear tobe (1) of bacterial origin, (2) present in the last eukaryote com-mon ancestor (LECA) (universal or nearly universal amongeukaryotes), and (3) with strong phylogenetic signal (out-paralog free and consistent with well-supported eukaryotephylogeny [5]). Of the 281 universal euBac proteins identiﬁed,most failed the latter criteria, primarily due to early gene dupli-cation and lineage-speciﬁc losses.Thirty-seven euBacs survived all screening protocols, 33 ofwhichareknownorpredictedtofunctioninthemitochondrion(Table S1). To increase sampling for Excavata, we sequencedthe Acrasis kona (Heterolobosea, Discoba) transcriptome,yieldingafullsetof37euBacproteins.Outgrouptaxaincludedthe closest bacterial relatives of the 37 euBacs (Table S2). AlleuBacs in the ﬁnal data set reproduce eight or more of theten major eukaryote groups represented, and 36 euBacsshow >60% maximum-likelihood bootstrap (mlBP) supportfor six or more of these major groups (Table S4).A Rooted Phylogeny of EukaryotesPhylogenetic analysis of a concatenation of the 37 euBacprotein sequences produces a highly resolved phylogeny ofeukaryotes (Figure 1). All major nodes are strongly supportedbased on mlBP [8] and Bayesian inference posterior proba-bilities (biPP) [9]. All ten major eukaryote clades representedare reproduced as monophyletic (1.0/100% biPP/mlBP;Figure 1). These major clades are further organized into threemegagroups (1.0/98%–100% biPP/mlBP; Figure 1): Discoba(Excavata), Amorphea (formerly Unikonta), and SA[R]P(Stramenopila+Alveolata+[Rhizarianotrepresented]+Plantae).Among these, Amorphea and SA[R]P form a clade to theexclusion of Discoba (1.0/90% biPP/mlBP; Figure 1, matrixM). This identiﬁes Discoba, and potentially its parent taxonExcavata, as the sister lineage to the bulk of knowneukaryote diversity (‘‘Neozoa’’ [6]), thus yielding a ‘‘neozoan-excavate’’ root.Amajorconcerninarootedphylogenyispossibleartifactualattractionoflongingroupbranchestowardthepotentiallylongbranches of a distant outgroup (long-branch attraction, orLBA)[10].WetestedtheeuBacphylogenyforpossiblesourcesof LBA, particularly signal dependence on fast-evolving align-ment positions and distant outgroup sequences. Removal ofthe fastest-evolving alignment positions (3,017 sites, or 21%of the data set), deﬁned in a tree-independent manner [11],yielded an identical maximum-likelihood tree with strongsupport for the neozoan-excavate root (0.99/92% biPP/mlBP; Figure 1, matrix M

Report An Alternative Root for the Eukaryote Tree of Life

https://www.cell.com/current-biology/pdf/S0960-9822(14)00486-2.pdf

Transcriptional regulation by Pho23 modulates the frequency of autophagosome formation.

Optical and molecular signatures of dissolved organic matter in Xiangxi Bay and mainstream of Three Gorges Reservoir, China: Spatial variations and environmental implications

Ding He

Papers

The machinery of macroautophagy

An Alternative Root for the Eukaryote Tree of Life

Report An Alternative Root for the Eukaryote Tree of Life

Transcriptional regulation by Pho23 modulates the frequency of autophagosome formation.

Optical and molecular signatures of dissolved organic matter in Xiangxi Bay and mainstream of Three Gorges Reservoir, China: Spatial variations and environmental implications