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

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 fl ux 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, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost 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 fi eld 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 delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so 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 autophagy assays, we hope to encourage technical innovation in the field

Cadherin evolution and the origin of animals

The question of how animals evolved from a unicellular ancestor has challenged evolutionary biologists for decades. Because cell adhesion and signaling are required for multicellularity, understanding how these cellular processes evolved will provide key insights into the origin of animals. A critical finding is that choanoflagellates, the closest living unicellular relatives of animals, express members of the cadherin superfamily. Cadherins are pivotal for animal cell adhesion and signaling and were previously thought to be unique to animals, making them crucial to understanding the evolutionary origin and transition to multicellularity. Importantly, the presence of cadherins in choanoflagellates allows a consideration of their ancestral function in the unicellular progenitor of animals. To gain insight into the ancestral structure and function of cadherins, I reconstructed the domain content of cadherins from the last common ancestor of choanoflagellates and metazoans. Conservation of diverse protein domains in the choanoflagellate Monosiga brevicollis and metazoan cadherins suggests that ancestral cadherins served both signaling and adhesive functions. I find that two M. brevicollis cadherin containing an ancestral domain combination MBCDH1 and a close paralog, MBCDH2, localize to the actin-filled microvilli of the feeding collar. Interestingly, the protein abundance of these cadherins changes in response to bacterial food availability. In vitro studies, as well as experiments performed in a heterologous cell culture system, suggest that MBCDH1 does not mediate homophilic adhesion in the context of these experiments like some metazoan cadherins and thus may play a role in cell signaling. Taken together, these data suggest that cadherins may mediate interactions with the extracellular environment, including the recognition and capture of bacterial prey. I hypothesize that metazoan cadherins were co-opted to mediate cell-cell interactions from ancestral proteins that interpreted and responded to extracellular cues

Predicted glycosyltransferases promote development and prevent spurious cell clumping in the choanoflagellate S. rosetta.

In a previous study we established forward genetics in the choanoflagellate Salpingoeca rosetta and found that a C-type lectin gene is required for rosette development (Levin et al., 2014). Here we report on critical improvements to genetic screens in S. rosetta while also investigating the genetic basis for rosette defect mutants in which single cells fail to develop into orderly rosettes and instead aggregate promiscuously into amorphous clumps of cells. Two of the mutants, Jumble and Couscous, mapped to lesions in genes encoding two different predicted glycosyltransferases and displayed aberrant glycosylation patterns in the basal extracellular matrix (ECM). In animals, glycosyltransferases sculpt the polysaccharide-rich ECM, regulate integrin and cadherin activity, and, when disrupted, contribute to tumorigenesis. The finding that predicted glycosyltransferases promote proper rosette development and prevent cell aggregation in S. rosetta suggests a pre-metazoan role for glycosyltransferases in regulating development and preventing abnormal tumor-like multicellularity

Predicted glycosyltransferases promote development and prevent spurious cell clumping in the choanoflagellate S. rosetta.

In a previous study we established forward genetics in the choanoflagellate Salpingoeca rosetta and found that a C-type lectin gene is required for rosette development (Levin et al., 2014). Here we report on critical improvements to genetic screens in S. rosetta while also investigating the genetic basis for rosette defect mutants in which single cells fail to develop into orderly rosettes and instead aggregate promiscuously into amorphous clumps of cells. Two of the mutants, Jumble and Couscous, mapped to lesions in genes encoding two different predicted glycosyltransferases and displayed aberrant glycosylation patterns in the basal extracellular matrix (ECM). In animals, glycosyltransferases sculpt the polysaccharide-rich ECM, regulate integrin and cadherin activity, and, when disrupted, contribute to tumorigenesis. The finding that predicted glycosyltransferases promote proper rosette development and prevent cell aggregation in S. rosetta suggests a pre-metazoan role for glycosyltransferases in regulating development and preventing abnormal tumor-like multicellularity

Cilia-associated oxysterols activate smoothened

Primary cilia are required for Smoothened to transduce vertebrate Hedgehog signals, but how Smoothened accumulates in cilia and is activated is incompletely understood. Here, we identify cilia-associated oxysterols that promote Smoothened accumulation in cilia and activate the Hedgehog pathway. Our data reveal that cilia-associated oxysterols bind to two distinct Smoothened domains to modulate Smoothened accumulation in cilia and tune the intensity of Hedgehog pathway activation. We find that the oxysterol synthase HSD11β2 participates in the production of Smoothened-activating oxysterols and promotes Hedgehog pathway activity. Inhibiting oxysterol biosynthesis impedes oncogenic Hedgehog pathway activation and attenuates the growth of Hedgehog pathway-associated medulloblastoma, suggesting that targeted inhibition of Smoothened-activating oxysterol production may be therapeutically useful for patients with Hedgehog-associated cancers.This work was supported by grants from the NIH (HL007731 and CA212279-01), the UCSF Physician Scientist Scholar Program, the American Society of Clinical Oncology, the Rally Foundation for Childhood Cancer Research, and the American Brain Tumor Association to D.R.R.; the NIH program Project Grant to Molecular Genetics (HL20948) to J.G.M.; Cancer Research UK (C20724 and A14414) and the European Research Council (647278) to C.S.; the NIH (AR065409 and HD092659) to S.Y.W. and L.X., respectively; the NIH (R01GM102498), the Ludwig Cancer Institute, and the Howard Hughes Medical Institute to P.A.B.; and the NIH (AR054396 and GM095941), the Burroughs Wellcome Fund, and the Packard Foundation to J.F.R.Peer reviewe

Erratum to: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (Autophagy, 12, 1, 1-222, 10.1080/15548627.2015.1100356

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