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)

Lipid droplets are storage organelles at the centre of lipid and energy homeostasis. They have a unique architecture consisting of a hydrophobic core of neutral lipids, which is enclosed by a phospholipid monolayer that is decorated by a specific set of proteins. Originating from the endoplasmic reticulum, lipid droplets can associate with most other cellular organelles through membrane contact sites. It is becoming apparent that these contacts between lipid droplets and other organelles are highly dynamic and coupled to the cycles of lipid droplet expansion and shrinkage. Importantly, lipid droplet biogenesis and degradation, as well as their interactions with other organelles, are tightly coupled to cellular metabolism and are critical to buffer the levels of toxic lipid species. Thus, lipid droplets facilitate the coordination and communication between different organelles and act as vital hubs of cellular metabolism.

Dynamics and functions of lipid droplets

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)

Over the past two decades, the molecular machinery that underlies autophagic responses has been characterized with ever increasing precision in multiple model organisms. Moreover, it has become clear that autophagy and autophagy-related processes have profound implications for human pathophysiology. However, considerable confusion persists about the use of appropriate terms to indicate specific types of autophagy and some components of the autophagy machinery, which may have detrimental effects on the expansion of the field. Driven by the overt recognition of such a potential obstacle, a panel of leading experts in the field attempts here to define several autophagy-related terms based on specific biochemical features. The ultimate objective of this collaborative exchange is to formulate recommendations that facilitate the dissemination of knowledge within and outside the field of autophagy research.

/pdf/molecular-definitions-of-autophagy-and-related-processes-27en62okjr.pdf

Molecular definitions of autophagy and related processes

Lipid droplets (LDs) are ubiquitous organelles that store neutral lipids for energy or membrane synthesis and act as hubs for metabolic processes. Cells generate LDs de novo, converting cells to emulsions with LDs constituting the dispersed oil phase in the aqueous cytoplasm. Here we review our current view of LD biogenesis. We present a model of LD formation from the ER in distinct steps and highlight the biology of proteins that govern this biophysical process. Areas of incomplete knowledge are identified, as are connections with physiology and diseases linked to alterations in LD biology.

/pdf/lipid-droplet-biogenesis-4cvf140s0j.pdf

Lipid Droplet Biogenesis.

Stationary phase (stat-phase) is a poorly understood physiological state under which cells arrest proliferation and acquire resistance to multiple stresses Lipid droplets (LDs), organelles specialized for cellular lipid homeostasis, increase in size and number at the onset of stat-phase However, little is known about the dynamics of LDs under this condition In this paper, we reveal the passage of LDs from perinuclear endoplasmic reticulum association to entry into vacuoles during the transition to stat-phase We show that the process requires the core autophagy machinery and a subset of autophagy-related (Atg) proteins involved in selective autophagy Notably, the process that we term stat-phase lipophagy is mediated through a sterol-enriched vacuolar microdomain whose formation and integrity directly affect LD translocation Intriguingly, cells defective in stat-phase lipophagy showed disrupted vacuolar microdomains, implying that LD contents, likely sterol esters, contribute to the maintenance of vacuolar microdomains Together, we propose a feed-forward loop in which lipophagy stimulates vacuolar microdomain formation, which in turn promotes lipophagy during stat-phase

https://rupress.org/jcb/article-pdf/206/3/357/1364819/jcb_201404115.pdf

A sterol-enriched vacuolar microdomain mediates stationary phase lipophagy in budding yeast

The human congenital generalized lipodystrophy type 2 protein seipin (Fld1 in budding yeast) controls lipid droplet (LD) size through an unknown mechanism. Here, we report that deletion of yeast LDB16/YCL005W , similar to deletion of FLD1 , causes supersized and small clustered LDs, altered phospholipid metabolism and impaired distribution of a subset of LD proteins. Ldb16 is a transmembrane protein in the endoplasmic reticulum (ER) that assembles together with Fld1 at ER–LD contact sites, a region that probably links neutral lipid synthesis with LD assembly. The formation of the Fld1–Ldb16 complex involves putative transmembrane segments of both proteins, thus, directly contributing to the maintenance of LD morphology. The stability of Ldb16 requires Fld1, as Ldb16 is subjected to ER-associated degradation (ERAD) in the absence of Fld1 but is stabilized when Fld1 is present. Strikingly, human seipin, but not yeast Fld1, complements the defects in LDs in ldb16Δ yeast, implying that seipin can substitute for the function of the Fld1–Ldb16 complex. We propose that human seipin might adopt the architecture of the yeast Fld1–Ldb16 complex in order to properly maintain the size of LDs.

/pdf/control-of-lipid-droplet-size-in-budding-yeast-requires-the-2yn9ffsye7.pdf

Control of lipid droplet size in budding yeast requires the collaboration between Fld1 and Ldb16.

This paper reviews the NTIRE 2022 challenge on efficient single image super-resolution with focus on the proposed solutions and results. The task of the challenge was to super-resolve an input image with a magnification factor of ×4 based on pairs of low and corresponding high resolution images. The aim was to design a network for single image super-resolution that achieved improvement of efficiency measured according to several metrics including runtime, parameters, FLOPs, activations, and memory consumption while at least maintaining the PSNR of 29.00dB on DIV2K validation set. IMDN is set as the baseline for efficiency measurement. The challenge had 3 tracks including the main track (runtime), sub-track one (model complexity), and sub-track two (overall performance). In the main track, the practical runtime performance of the submissions was evaluated. The rank of the teams were determined directly by the absolute value of the average runtime on the validation set and test set. In sub-track one, the number of parameters and FLOPs were considered. And the individual rankings of the two metrics were summed up to determine a final ranking in this track. In sub-track two, all of the five metrics mentioned in the description of the challenge including runtime, parameter count, FLOPs, activations, and memory consumption were considered. Similar to sub-track one, the rankings of five metrics were summed up to determine a final ranking. The challenge had 303 registered participants, and 43 teams made valid submissions. They gauge the state-of-the-art in efficient single image super-resolution.

Yu-Hsuan Miao

Papers

A sterol-enriched vacuolar microdomain mediates stationary phase lipophagy in budding yeast

Control of lipid droplet size in budding yeast requires the collaboration between Fld1 and Ldb16.

NTIRE 2022 Challenge on Efficient Super-Resolution: Methods and Results