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

Great progress has been made toward understanding the pathogenesis of Parkinson's disease (PD) during the past two decades, mainly as a consequence of the discovery of specific gene mutations contributing to the onset of PD. Recently, dysregulation of the autophagy pathway has been observed in the brains of PD patients and in animal models of PD, indicating the emerging role of autophagy in this disease. Indeed, autophagy is increasingly implicated in a number of pathophysiologies, including various neurodegenerative diseases. This article will lead you through the connection between autophagy and PD by introducing the concept and physiological function of autophagy, and the proteins related to autosomal dominant and autosomal recessive PD, particularly a-synuclein and PINK1-PARKIN, as they pertain to autophagy.T here seem to be various causes of Parkinson's disease (PD), yet the pathogenesis of this disease appears to be converging on common themes-oxidative stress, mitochondrial dysfunction, and protein aggregation-all of which are tightly linked to autophagy, a highly conserved cellular homeostatic process essential for bulk degradation of cytoplasmic contents. In particular, the recent identification of autosomal dominant and autosomal recessive mutations in familial PD has revealed the involvement of the corresponding gene products in autophagy. Although autophagy has commonly been regarded as an adaptive response to nutrient deprivation, increasing evidence indicates that basal, constitutive autophagy is essential for neuronal survival and that its dysregulation leads to neurodegeneration. AUTOPHAGY Main Types of AutophagyAutophagy is an evolutionarily conserved catabolic process that mediates the degradation of long-lived proteins and dysfunctional or superfluous organelles in eukaryotic cells. Autophagy is induced by various adverse conditions such as limited nutrients, low oxygen levels, and decreased energy supply, and its action results in the release of degradation products, especially amino acids, back into the cytoplasm to be used in essential biosynthetic pathways.According to the different pathways by which cargo is delivered to the lysosome or vacuole, autophagy can be divided into three main types (Fig. 1): macroautophagy, microautophagy, and chaperone-mediated autophagy

The ability to switch from skotomorphogenesis to photomorphogenesis is essential for seedling development and plant survival. Recent studies revealed that COP1 and phytochrome-interacting factors

The MAP kinase Slt2 is required for both mitophagy and pexophagy, whereas the MAP kinase Hog1 acts specifically in mitophagy.

Autophagy has been implicated in a number of physiological processes important for human heath and disease. Autophagy involves the formation of a double-membrane cytosolic vesicle, an autophagosome. Central to the formation of the autophagosome is the ubiquitin-like protein autophagy-related (Atg)8 (microtubuleassociated protein 1 light chain 3/LC3 in mammalian cells). Following autophagy induction, Atg8 shows the greatest change in expression of any of the proteins required for autophagy. The magnitude of autophagy is, in part, controlled by the amount of Atg8; thus, controlling Atg8 protein levels is one potential mechanism for modulating autophagy activity. We have identified a negative regulator of ATG8 transcription, Ume6, which acts along with a histone deacetylase complex including Sin3 and Rpd3 to regulate Atg8 levels; deletion of any of these components leads to an increase in Atg8 and a concomitant increase in autophagic activity. A similar regulatory mechanism is present in mammalian cells, indicating that this process is highly conserved.lysosome | membrane biogenesis | phagophore | stress | vacuole M acroautophagy, hereafter referred to as autophagy, is an evolutionarily conserved process used by eukaryotic cells for the bulk degradation of intracellular proteins and organelles (1). Autophagy is not only vital for cell survival in nutrient-poor conditions (2) but is also linked to various physiological processes, including immune defense, tumor suppression, and prevention of neurodegeneration (3). Whereas autophagy plays a primarily protective role, it can also contribute to cell death; thus, the magnitude of autophagy must be carefully regulated.Central to autophagy is the formation of autophagosomes (4), double-membrane-bound structures that engulf and deliver cytoplasmic materials to the vacuole/lysosome for degradation. During autophagosome formation, the autophagy-related ubiquitin-like protein Atg8/microtubule-associated protein 1 light chain 3 (LC3) covalently modifies phosphatidylethanolamine (PE). Almost one-fourth of the characterized autophagy-related (Atg) proteins in yeast are involved in the formation or stability of Atg8-PE, which plays a critical role in controlling expansion of the phagophore (the initial sequestering membrane), and in determining autophagosome size, thereby regulating autophagy activity (5, 6). Upon starvation, the level of ATG8 mRNA sharply increases, leading to a subsequent induction of the Atg8 protein level (7,8). The increase in the amount of Atg8 during autophagy is critical for supplying a sufficient amount of this protein to maintain normal levels of autophagy; yeast strains deficient in Atg8 induction generate abnormally small autophagosomes (5). Thus, characterization of how Atg8 protein levels are modulated is of tremendous importance both in understanding the regulation of autophagy and for the elucidation of potential therapeutic targets. However, little is known about the mechanisms regulating ATG8 transcription. ResultsUme6-Sin3-Rpd3 Complex Represse...

Summary Background The biogenesis of autophagosomes, the hallmark of autophagy, depends on the function of the autophagy-related (Atg) proteins and the generation of phosphatidylinositol-3-phosphate (PtdIns3P) at the phagophore assembly site (PAS), the location where autophagosomes arise. The current model is that PtdIns3P is involved primarily in the recruitment of Atg proteins to the PAS and that once an autophagosome is complete, the Atg machinery is released from its surface back into the cytoplasm and reused for the formation of new vesicles. Results We have identified a PtdIns3P phosphatase, Ymr1, that is essential for the normal progression of both bulk and selective types of autophagy. This protein is recruited to the PAS at an early stage of formation of this structure through a process that requires both its GRAM domain and its catalytic activity. In the absence of Ymr1, Atg proteins fail to dissociate from the limiting membrane of autophagosomes, and these vesicles accumulate in the cytoplasm. Conclusions Our data thus reveal a key role for PtdIns3P turnover in the regulation of the late steps of autophagosome biogenesis and indicate that the disassembly of the Atg machinery from the surface of autophagosomes is a requisite for their fusion with the vacuole.

AMP-activated protein kinase (AMPK) is a highly conserved cellular energy sensor that plays a central role in metabolic homeostasis. A recent study in Science (Egan et al., 2011) identifies ULK1 as a substrate for AMPK phosphorylation, a modification required for selective autophagy of mitochondria and cell survival during starvation.

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