Chapter Twenty‐Eight Qualitative and Quantitative Characterization of Autophagy in Caenorhabditis elegans by Electron Microscopy

Sigmond, Tímea; Fehér, Judit; Baksa, Attila; Pásti, Gabriella; Pálfia, Zsolt; Takács‐Vellai, Krisztina; Kovács, János; Vellai, Tibor; Kovács, Attila

doi:10.1016/s0076-6879(08)03228-x

Cited by 23 publications

(35 citation statements)

References 22 publications

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“…45,46,65,66 Care in the choice of sample to be analyzed is critical to the success of TEM studies for autophagy. Whereas fixation of in vitro samples is relatively straightforward, fixation of excised tissues requires care to avoid sampling a nonrepresentative or uninformative section of tissue.…”

Section: Introductionmentioning

confidence: 99%

“…The best approach is to estimate the volume occupied by autophagic structures (as percent of cytoplasmic or cellular volume, using volumetric morphometry/stereology) in at least 20 cell profiles per sample (the number needed should be dictated by some form of power analysis, which indicates that the data are significant). 20,65,68,70,71 During quantification it is important to make sure that each imaged cell profile is captured and scored at the same magnification, and that every cell profile in the thin section has an equal probability to be included in the counting.…”

Section: Introductionmentioning

confidence: 99%

“…Thus, double membranes cannot be relied upon as the sole means for the ultrastructural identification of autophagyrelated structures, and it is important to employ expert analysis to avoid misinterpretation of micrographs. 46,65,66 In some cases, it may be prudent to employ tomographic reconstructions of the TEM images to confirm that the autophagic compartments are spherical and are not being confused with endomembrane cisternae or damaged mitochondria with similar appearance in thin-sections (e.g., see ref. 73), but this is obviously a timeconsuming approach requiring sophisticated equipment.…”

Section: Introductionmentioning

confidence: 99%

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Guidelines for the use and interpretation of assays for monitoring autophagy

Klionsky¹,

Abdalla²,

Abeliovich³

et al. 2012

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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

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Guidelines for the use and interpretation of assays for monitoring autophagy

Klionsky¹,

Abdalla²,

Abeliovich³

et al. 2012

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“…Care must also be exercised to exclude the false recognition of endocytosed apoptotic bodies or interdigitations of the plasma membrane, as well as of deteriorated or swollen mitochondria or occasional secretory vacuoles with heterogeneous contents. 12,18,22,233,234 Obviously, the use of IEM to detect specific protein markers can circumvent these difficulties in the reliable identification of autophagic compartments.…”

Section: Remarks On Autophagosome Identification and Quantification Imentioning

confidence: 99%

Seeing is believing: The impact of electron microscopy on autophagy research

et al. 2011

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Autophagy was first discovered by transmission electron microscopy more than 50 years ago. For decades, electron microscopy was the only way to reliably detect autophagic compartments in cells because no specific protein markers were known. In the 1970s, however, the introduction of biochemical methods enabled quantitative studies of autophagic-lysosomal degradation, and in the 1980s specific biochemical assays for autophagic sequestration became available. Since the identification of autophagy-related genes in the 1990s, combined fluorescence microscopy, biochemical and genetic methods have taken the leading role in autophagy research. However, electron microscopy is still needed to confirm and verify results obtained by other methods, and also to produce novel knowledge that would not be achievable by any other experimental approach. Confocal microscopy, with its ever-improving resolution, is probably the best-suited morphological approach to investigate the dynamic aspects of autophagy. However, for analyzing the ultrastructural details of the many novel organelles and mechanisms involved in specific subtypes of autophagy, the electron microscope is still indispensable. This review will summarize the impact that electron microscopy has had on autophagy research since the discovery of this self-degradation process in the mid-1950s. Astonishingly, some of the "novel" concepts and principles of autophagy, presented in the recent studies, were already proposed several decades ago by the pioneering, accurate and passionate work of virtuoso electron microscopists.

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“…3,6 The problems in the identification of autophagic compartments were well known to the early electron microscopists, who described the macroautophagic process and explored its details in the 1960s, 1970s and 1980s. The quality of the electron microscopy images published in those early studies is generally very high, and the images have also been interpreted carefully and correctly.…”

Section: O N O T D I S T R I B U T Ementioning

confidence: 99%