TY - JOUR
T1 - Guidelines for the use and interpretation of assays for monitoring autophagy
AU - Klionsky, Daniel J.
AU - Abdalla, Fabio C.
AU - Abeliovich, Hagai
AU - Abraham, Robert T.
AU - Acevedo-Arozena, Abraham
AU - Adeli, Khosrow
AU - Agholme, Lotta
AU - Agnello, Maria
AU - Agostinis, Patrizia
AU - Aguirre-Ghiso, Julio A.
AU - Ahn, Hyung Jun
AU - Ait-Mohamed, Ouardia
AU - Ait-Si-Ali, Slimane
AU - Akematsu, Takahiko
AU - Akira, Shizuo
AU - Al-Younes, Hesham M.
AU - Al-Zeer, Munir A.
AU - Albert, Matthew L.
AU - Albin, Roger L.
AU - Alegre-Abarrategui, Javier
AU - Aleo, Maria Francesca
AU - Alirezaei, Mehrdad
AU - Almasan, Alexandru
AU - Almonte-Becerril, Maylin
AU - Amano, Atsuo
AU - Amaravadi, Ravi
AU - Amarnath, Shoba
AU - Amer, Amal O.
AU - Andrieu-Abadie, Nathalie
AU - Anantharam, Vellareddy
AU - Ann, David K.
AU - Anoopkumar-Dukie, Shailendra
AU - Aoki, Hiroshi
AU - Apostolova, Nadezda
AU - Arancia, Giuseppe
AU - Aris, John P.
AU - Asanuma, Katsuhiko
AU - Asare, Nana Y. O.
AU - Ashida, Hisashi
AU - Askanas, Valerie
AU - Askew, David S.
AU - Auberger, Patrick
AU - Baba, Misuzu
AU - Backues, Steven K.
AU - Baehrecke, Eric H.
AU - Bahr, Ben A.
AU - Bai, Xue-Yuan
AU - Bailly, Yannick
AU - Baiocchi, Robert
AU - Rouschop, Kasper M. A.
AU - Author collaboration
PY - 2012/4
Y1 - 2012/4
N2 - 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.
AB - 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.
KW - Lc3
KW - Autolysosome
KW - Autophagosome
KW - Flux
KW - Lysosome
KW - Phagophore
KW - Stress
KW - Vacuole
U2 - 10.4161/auto.19496
DO - 10.4161/auto.19496
M3 - (Systematic) Review article
C2 - 22966490
SN - 1554-8627
VL - 8
SP - 445
EP - 544
JO - Autophagy
JF - Autophagy
IS - 4
ER -