Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)
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Abstract
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 thatthere 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 completeprocess 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 increasedautophagy 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 field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in manycases, 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 forreviewers 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 multipleassays 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 autophagyrelated 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.
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The record
- Venue
- Autophagy
- Topic
- Autophagy in Disease and Therapy
- Field
- Medicine
- Canadian institutions
- Libin Cardiovascular Institute of AlbertaSunnybrook HospitalUniversity of CalgaryConcordia UniversityUniversity of WaterlooCanada's Michael Smith Genome Sciences CentreTerry Fox Research InstituteInstitute for Research in Immunology and CancerManitoba HealthYork UniversityInnovation and Economic Development Trois RivièresBC Cancer AgencyUniversity Health NetworkUniversity of AlbertaUniversity of British ColumbiaCancerCare ManitobaUniversity of ManitobaPrevention of Organ FailureUniversité LavalCentre hospitalier universitaire de QuébecJewish General HospitalSimon Fraser UniversityDalhousie UniversityUniversity of OttawaMcGill UniversityUniversité du Québec à Trois-RivièresMcGill University Health CentreLunenfeld-Tanenbaum Research InstituteSt. Paul's HospitalWestern UniversityHospital for Sick ChildrenRoyal Military College of CanadaUniversité de MontréalMontreal Neurological Institute and HospitalUniversité de SherbrookeUniversité du Québec à MontréalMcMaster UniversityUniversity of TorontoSunnybrook Health Science CentreInstitute of Infection and Immunity
- Funders
- Eunice Kennedy Shriver National Institute of Child Health and Human DevelopmentNational Institute on Minority Health and Health DisparitiesNational Institute of Environmental Health SciencesNational Institute of Arthritis and Musculoskeletal and Skin DiseasesNational Institute of Diabetes and Digestive and Kidney DiseasesNational Institute on Drug AbuseNational Heart, Lung, and Blood InstituteNational Institute on Alcohol Abuse and AlcoholismNational Eye InstituteNational Center for Complementary and Integrative HealthNational Institute for Health and Care ResearchNational Institute on AgingNational Cancer InstituteWellcome TrustCancer Research UKBiotechnology and Biological Sciences Research CouncilNational Center for Research ResourcesNational Institute of Allergy and Infectious DiseasesMedical Research CouncilBrain Tumour CharityNational Institutes of HealthU.S. Department of Veterans AffairsSchweizerischer Nationalfonds zur Förderung der Wissenschaftlichen ForschungNovo Nordisk FondenDoris Duke Charitable FoundationNational Institute of Neurological Disorders and StrokeNational Centre for the Replacement, Refinement and Reduction of Animals in ResearchParkinson's UKLundbeckfondenAction Medical ResearchNational Institute of General Medical Sciences
- Keywords
- AutophagyAutophagosomeBiologyCell biologyVacuolePhagosomeATG8Multicellular organismBiogenesisIntracellularCellBiochemistryCytoplasm
- Has abstract in OpenAlex
- yes