Estimating the size and number of autophagic bodies by electron microscopy

Backues, Steven K.; Chen, Dachuan; Ruan, Jishou; Xie, Zhiping; Klionsky, Daniel J.

doi:10.4161/auto.26856

Cited by 57 publications

(71 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3A); the size and number of autophagic bodies were quantified and estimated as described previously. 21 The average size of the autophagic bodies in both mutants was not significantly different compared to the WT (Fig. S2).…”

Section: Phosphorylation Of Atg9 S122 Is Important For Selective Automentioning

confidence: 93%

“…Finally, our data suggest that the S122D mutation results in an enhanced interaction in particular between Atg9 and Atg27, one of the components required for efficient anterograde trafficking of Atg9. 21,45 We note, however, that Atg23 and Atg27 do not appear to be conserved in higher eukaryotes, so different components my take the place of these proteins if this regulatory aspect of Atg9 trafficking is conserved beyond fungi.…”

Section: Discussionmentioning

confidence: 99%

“…21,49,57,58 Antibodies against Atg9, Ape1 and Pgk1 (a generous gift from Dr. Jeremy Thorner, University of California, Berkeley), and a commercial antibody that reacts with protein A (no longer available) were used as described previously. 15,35 Anti-Dpm1 was from Molecular Probes/Invitrogen (A-6429).…”

Section: Additional Assaysmentioning

confidence: 99%

“…18 Thus, Atg9 may move between these peripheral sites and the PAS, and earlier studies have suggested that this cycling of Atg9 is required for autophagosome formation, functioning in some manner to direct membrane to the expanding phagophore. 16,[19][20][21] In mammalian cells, ATG9 also displays dynamic cycling, moving between the trans-Golgi network and endosomes in response to nutritional changes. A subpopulation of ATG9 colocalizes with both LC3, a mammalian homolog of Atg8, and RAB7, a late endosomal protein.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Phosphorylation of Atg9 regulates movement to the phagophore assembly site and the rate of autophagosome formation

Feng¹,

Backues²,

Baba³

et al. 2016

Autophagy

View full text Add to dashboard Cite

Macroautophagy is primarily a degradative process that cells use to break down their own components to recycle macromolecules and provide energy under stress conditions, and defects in macroautophagy lead to a wide range of diseases. Atg9, conserved from yeast to mammals, is the only identified transmembrane protein in the yeast core macroautophagy machinery required for formation of the sequestering compartment termed the autophagosome. This protein undergoes dynamic movement between the phagophore assembly site (PAS), where the autophagosome precursor is nucleated, and peripheral sites that may provide donor membrane for expansion of the phagophore. Atg9 is a phosphoprotein that is regulated by the Atg1 kinase. We used stable isotope labeling by amino acids in cell culture (SILAC) to identify phosphorylation sites on this protein and identified an Atg1-independent phosphorylation site at serine 122. A nonphosphorylatable Atg9 mutant showed decreased autophagy activity, whereas the phosphomimetic mutant enhanced activity. Electron microscopy analysis suggests that the different levels of autophagy activity reflect differences in autophagosome formation, correlating with the delivery of Atg9 to the PAS. Finally, this phosphorylation regulates Atg9 interaction with Atg23 and Atg27.

show abstract

Section: Phosphorylation Of Atg9 S122 Is Important For Selective Automentioning

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Section: Additional Assaysmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Phosphorylation of Atg9 regulates movement to the phagophore assembly site and the rate of autophagosome formation

Feng¹,

Backues²,

Baba³

et al. 2016

Autophagy

View full text Add to dashboard Cite

show abstract

“…The main function of autophagy is to recycle damaged or old cellular constituents by encapsulating cargo within autophagosomes that are then trafficked to fuse with lysosomes, allowing lysosomal hydrolytic enzymes to catabolize the cellular material into simple macromolecular subunits. Induction of autophagy above basal levels provided some of the first insights into the complex role of this dynamic process in cell death and its role in radiation sensitization [4,5].To provide a proof-of-principle, morphomics data was collected and analyzed for human glioblastoma (U87) monolayer cultured cells irradiated with 0 or 10 Gy  rays [2] and 3 days after treatment were collected and fixed for TEM (the most sensitive method to monitor autophagy [6]). Biased sampling methods were used to selectively acquire at least 30 cell profiles for each treatment.…”

mentioning

confidence: 99%

Measurement of radiation-induced autophagic flux in human brain cancer cells

et al. 2015

View full text Add to dashboard Cite

Radiation-induced autophagy mediates susceptibility to radiation [1]. However, autophagy is a multifaceted process. Herein, 2 aspects of autophagy, induction of autophagy or changes in autophagic flux, in radiation sensitivity were investigated. Relying on a systems-based approach and adding to a large, data dense morphome database [2, 3], we mined for complex cellular information on autophagy due to irradiation of human brain cancer (glioblastoma or GBM) cells. A quantitative stereology method is described here to chronicle these two important aspects of autophagy. These results add to our understanding of the role of autophagy in the efficacy of radiation therapy by providing a basis for the adaptive role for radiation-induced autophagy in GBM cells. Further, because GBM tumors are so resistant to radiotherapy, a novel strategy to radiosensitize GBM cells is urgently needed.Autophagy is a normal, basal housekeeping process in a cell. The main function of autophagy is to recycle damaged or old cellular constituents by encapsulating cargo within autophagosomes that are then trafficked to fuse with lysosomes, allowing lysosomal hydrolytic enzymes to catabolize the cellular material into simple macromolecular subunits. Induction of autophagy above basal levels provided some of the first insights into the complex role of this dynamic process in cell death and its role in radiation sensitization [4,5].To provide a proof-of-principle, morphomics data was collected and analyzed for human glioblastoma (U87) monolayer cultured cells irradiated with 0 or 10 Gy  rays [2] and 3 days after treatment were collected and fixed for TEM (the most sensitive method to monitor autophagy [6]). Biased sampling methods were used to selectively acquire at least 30 cell profiles for each treatment. The number of autophagosomes was scored for each cell to reflect changes in induction of autophagy in the cell population and within each cell. Autophagic flux was estimated by measuring the area of the cytoplasm occupied by autophagosomes using ImageJ. The area measurement integrates both the number of autophagosomes and size of autophagosomes in the cell. Increased flux, a possible outcome of the area analysis, is revealed when a decreased number and area of autophagosomes is found because hydrolytic digestion of autophagosomes indicates completion of the autophagic process. In contrast, decreased autophagic flux is revealed by increased number or area of autophagosomes resulting from a blockade in autophagy before the final hydrolysis of autophagosomes. Finally, a third outcome of the analysis is the identification of cells that have cytoplasmic areas occupied by autophagosomes that are more than 1 standard deviation of the average area. These outliers focus our attention on those cells having the most dramatic change in radiation-induced autophagy where flux is decreased AND continued autophagosome formation is abnormal. Indeed, previous experiments identified a significant proportion of an irradiated population as outliers, having extre...

show abstract

Phagophore closure, autophagosome maturation and autophagosome fusion during macroautophagy in the yeast Saccharomyces cerevisiae

Kraft

Reggiori

2023

FEBS Letters

View full text Add to dashboard Cite

Macroautophagy, hereafter referred to as autophagy, is a complex process in which multiple membrane‐remodeling events lead to the formation of a cisterna known as the phagophore, which then expands and closes into a double‐membrane vesicle termed the autophagosome. During the past decade, enormous progress has been made in understanding the molecular function of the autophagy‐related proteins and their role in generating these phagophores. In this Review, we discuss the current understanding of three membrane remodeling steps in autophagy that remain to be largely characterized; namely, the closure of phagophores, the maturation of the resulting autophagosomes into fusion‐competent vesicles, and their fusion with vacuoles/lysosomes. Our review will mainly focus on the yeast Saccharomyces cerevisiae, which has been the leading model system for the study of molecular events in autophagy and has led to the discovery of the major mechanistic concepts, which have been found to be mostly conserved in higher eukaryotes.

show abstract

Estimating the size and number of autophagic bodies by electron microscopy

Cited by 57 publications

References 12 publications

Phosphorylation of Atg9 regulates movement to the phagophore assembly site and the rate of autophagosome formation

Phosphorylation of Atg9 regulates movement to the phagophore assembly site and the rate of autophagosome formation

Measurement of radiation-induced autophagic flux in human brain cancer cells

Phagophore closure, autophagosome maturation and autophagosome fusion during macroautophagy in the yeast Saccharomyces cerevisiae

Contact Info

Product

Resources

About