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)

Mitophagy and Quality Control Mechanisms in Mitochondrial Maintenance.

Mitophagy is an evolutionarily conserved cellular process to remove dysfunctional or superfluous mitochondria, thus fine-tuning mitochondrial number and preserving energy metabolism. In this Review, we survey recent advances towards elucidating the molecular mechanisms that mediate mitochondrial elimination and the signalling pathways that govern mitophagy. We consider the contributions of mitophagy in physiological and pathological contexts and discuss emerging findings, highlighting the potential value of mitophagy modulation in therapeutic intervention.

Mechanisms of mitophagy in cellular homeostasis, physiology and pathology.

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.

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Cargo recognition and degradation by selective autophagy

Mitochondria are essential for the viability of eukaryotic cells as they perform crucial functions in bioenergetics, metabolism and signalling and have been associated with numerous diseases. Recent functional and proteomic studies have revealed the remarkable complexity of mitochondrial protein organization. Protein machineries with diverse functions such as protein translocation, respiration, metabolite transport, protein quality control and the control of membrane architecture interact with each other in dynamic networks. In this Review, we discuss the emerging role of the mitochondrial protein import machinery as a key organizer of these mitochondrial protein networks. The preprotein translocases that reside on the mitochondrial membranes not only function during organelle biogenesis to deliver newly synthesized proteins to their final mitochondrial destination but also cooperate with numerous other mitochondrial protein complexes that perform a wide range of functions. Moreover, these protein networks form membrane contact sites, for example, with the endoplasmic reticulum, that are key for integration of mitochondria with cellular function, and defects in protein import can lead to diseases.

Mitochondrial proteins: from biogenesis to functional networks

Mitochondria are considered autonomous organelles, physically separated from endocytic and biosynthetic pathways. However, recent work uncovered a PINK1/parkin-dependent vesicle transport pathway wherein oxidized or damaged mitochondrial content are selectively delivered to the late endosome/lysosome for degradation, providing evidence that mitochondria are indeed integrated within the endomembrane system. Given that mitochondria have not been shown to use canonical soluble NSF attachment protein receptor (SNARE) machinery for fusion, the mechanism by which mitochondrial-derived vesicles (MDVs) are targeted to the endosomal compartment has remained unclear. In this study, we identify syntaxin-17 as a core mitochondrial SNARE required for the delivery of stress-induced PINK1/parkin-dependent MDVs to the late endosome/lysosome. Syntaxin-17 remains associated with mature MDVs and forms a ternary SNARE complex with SNAP29 and VAMP7 to mediate MDV-endolysosome fusion in a manner dependent on the homotypic fusion and vacuole protein sorting (HOPS) tethering complex. Syntaxin-17 can be traced to the last eukaryotic common ancestor, hinting that the removal of damaged mitochondrial content may represent one of the earliest vesicle transport routes in the cell.

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Sydney A. Lee

Papers

Syntaxin-17 delivers PINK1/parkin-dependent mitochondrial vesicles to the endolysosomal system