Guidelines for the use and interpretation of assays for monitoring autophagy

Klionsky, D. J. et al. (2014) Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy, 8(4), pp. 445-544. (doi:10.4161/auto.19496) (PMID:22966490) (PMCID:PMC3404883)

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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. 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.

Item Type:Articles
Additional Information:This work was supported in part by the National Institutes of Health, including Public Health Service grant GM053396 to D.J.K. Due to space and other limitations, it is not possible to include all other sources of financial support.
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Muller, Professor Sylke and King, Dr Jason and Holyoake, Professor Tessa and Helgason, Dr Vignir and Ryan, Professor Kevin and Mottram, Professor Jeremy
Authors: Klionsky, D. J., Abdalla, F. C., Abeliovich, H., Abraham, R. T., Acevedo-Arozena, A., Adeli, K., Agholme, L., Agnello, M., Agostinis, P., Aguirre-Ghiso, J. A., Ahn, H. J., Ait-Mohamed, O., Ait-Si-Ali, S., Akematsu, T., Akira, S., Al-Younes, H. M., Al-Zeer, M. A., Albert, M. L., Albin, R. L., Alegre-Abarrategui, J., Aleo, M. F., Alirezaei, M., Almasan, A., Almonte-Becerril, M., Amano, A., Amaravadi, R. K., Amarnath, S., Amer, A. O., Andrieu-Abadie, N., Anantharam, V., Ann, D. K., Anoopkumar-Dukie, S., Aoki, H., Apostolova, N., Arancia, G., Aris, J. P., Asanuma, K., Asare, N. Y.O., Ashida, H., Askanas, V., Askew, D. S., Auberger, P., Baba, M., Backues, S. K., Baehrecke, E. H., Bahr, B. A., Bai, X.-Y., Bailly, Y., Baiocchi, R., Baldini, G., Balduini, W., Ballabio, A., Bamber, B. A., Bampton, E. T.W., B´nhegyi, G.´b., Bartholomew, C. R., Bassham, D. C., Bast, R. C., Batoko, H., Bay, B.-H., Beau, I., Be´chet, D. M., Begley, T. J., Behl, C., Behrends, C., Bekri, S., Bellaire, B., Bendall, L. J., Benetti, L., Berliocchi, L., Bernardi, H., Bernassola, F., Besteiro, S.´b., Bhatia-Kissova, I., Bi, X., Biard-Piechaczyk, M., Blum, J. S., Boise, L. H., Bonaldo, P., Boone, D. L., Bornhauser, B. C., Bortoluci, K. R., Bossis, I., Bost, F.´d.´r., Bourquin, J.-P., Boya, P., Boyer-Guittaut, M., Bozhkov, P. V., Brady, N. R., Brancolini, C., Brech, A., Brenman, J. E., Brennand, A., Bresnick, E. H., Brest, P., Bridges, D., Bristol, M. L., Brookes, P. S., Brown, E. J., Brumell, J. H., Brunetti-Pierri, N., Brunk, U. T., Bulman, D. E., Bultman, S. J., Bultynck, G., Burbulla, L. F., Bursch, W., Butchar, J. P., Buzgariu, W., Bydlowski, S. P., Cadwell, K., Caho, M., Cai, D., Cai, J., Cai, Q., Calabretta, B., Calvo-Garrido, J., Camougrand, N., Campanella, M., Campos-Salinas, J., Candi, E., Cao, L., Caplan, A. B., Carding, S. R., Cardoso, S. M., Carew, J. S., Carlin, C. R., Carmignac, V., Carneiro, L. A.M., Carra, S., Caruso, R. A., Casari, G., Casas, C., Castino, R., Cebollero, E., Cecconi, F., Celli, J., Chaachouay, H., Chae, H.-J., Chai, C.-Y., Chan, D. C., Chan, E. Y., Chang, R. C.-C., Che, C.-M., Chen, C.-C., Chen, G.-C., Chen, G.-Q., Chen, M., Chen, Q., Chen, S. S.-L., Chen, W., Chen, X., Chen, X., Chen, X., Chen, Y.-G., Chen, Y., Chen, Y., Chen, Y.-J., Chen, Z., Cheng, A., Cheng, C. H.K., Cheng, Y., Cheong, H., Cheong, J.-H., Cherry, S., Chess-Williams, R., Cheung, Z. H., Chevet, E., Chiang, H.-L., Chiarelli, R., Chiba, T., Chin, L.-S., Chiou, S.-H., Chisari, F. V., Cho, C. H., Cho, D.-H., Choi, A. M.K., Choi, D., Choi, K. S., Choi, M. E., Chouaib, S., Choubey, D., Choubey, V., Chu, C. T., Chuang, T.-H., Chueh, S.-H., Chun, T., Chwae, Y.-J., Chye, M.-L., Ciarcia, R., Ciriolo, M. R., Clague, M. J., Clark, R. S.B., Clarke, P. G.H., Clarke, R., Codogno, P., Coller, H. A., Colombo, M.´a. I., Comincini, S., Condello, M., Condorelli, F., Cookson, M. R., Coombs, G. 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College/School:College of Medical Veterinary and Life Sciences > Institute of Cancer Sciences
Journal Name:Autophagy
Publisher:Landes Bioscience
ISSN:1554-8627
ISSN (Online):1554-8635
Published Online:01 April 2012

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
439231Is Bcr-Able expression relevant for the survival of cancer stem cells in chronic myeloid leukaemiaTessa HolyoakeMedical Research Council (MRC)G0600782RI CANCER SCIENCES