Atg13 and FIP200 act independently of Ulk1 and Ulk2 in autophagy induction

Alers, Sebastian; Löffler, Antje S.; Paasch, Florian; Dieterle, Alexandra; Keppeler, Hildegard; Lauber, Kirsten; Campbell, David G.; Fehrenbacher, Birgit; Schaller, Martin; Wesselborg, Sebastian; Stork, Björn

doi:10.4161/auto.7.12.18027

Cited by 119 publications

(134 citation statements)

References 39 publications

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“…Generally, the complete knockout of autophagy-regulatory proteins is preferable compared with RNAi-mediated knockdown, since in some cases these proteins function normally when expressed at reduced levels. 793 and autophagy can be analyzed by a variety of assays in this cell line. Steady-state methods that can be used include TEM, LC3 western blotting and fluorescence microscopy; flux measurements include monitoring LC3-II turnover and tandem mRFP/mCherry-GFP-LC3 fluorescence microscopy.…”

Section: 792mentioning

confidence: 99%

“…Using atg13 2/2 and ulk1/2 2/2 DT40 cells, it was shown that ATG13 and its binding capacity for RB1CC1 are mandatory for both basal and starvation-induced autophagy in this cell line, whereas ULK1/2 and in vitro-mapped ULK1-dependent phosphorylation sites of ATG13 appear to be dispensable for these processes. 793 Another useful system is chick retina, which can be used for monitoring autophagy at different stages of development. For example, lipidation of LC3 is observed during starvation, and can be blocked with a short-term incubation with 3-MA.…”

Section: 792mentioning

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: 792mentioning

confidence: 99%

Section: 792mentioning

confidence: 99%

Guidelines for the use and interpretation of assays for monitoring autophagy

Klionsky¹,

Abdalla²,

Abeliovich³

et al. 2012

Self Cite

View full text Add to dashboard Cite

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“…Finally, in Ulk1-silenced ulk2 -/-MEFs or in ulk1 -/-ulk2 -/-MEFs autophagy induced by amino acid deprivation is blocked, further supporting the redundant functions of both kinases [30,31]. Of note, starvation-induced autophagy was not inhibited in ulk1 -/-ulk2 -/-DT40 B lymphocytes generated in our laboratory, and ulk1 -/-ulk2 -/-MEFs display autophagy induction upon glucose deprivation [30,33]. The molecular details of these Ulk1/Ulk2-independent autophagy pathways have to be deciphered in the future.…”

Section: The Ulk1-atg13-fip200 Protein Kinase Complexmentioning

confidence: 80%

“…S48, T170, T331, T428 and T478 [33]. However, expression of the corresponding pan-serine/threonine-to-alanine mutant of Atg13 in atg13 -/-DT40 B cells did not block autophagy induction upon starvation [33]. Since Ulk1 and Ulk2 are dispensable for autophagy induction in this cellular system, potentially other kinases and Atg13 phospho-acceptor sites might play a role in the regulation of autophagy.…”

Section: The Ulk1-atg13-fip200 Protein Kinase Complex and Protein Phomentioning

confidence: 98%

“…Our group was able to identify five Ulk1-dependent in vitro phosphorylation sites in human Atg13, i.e. S48, T170, T331, T428 and T478 [33]. However, expression of the corresponding pan-serine/threonine-to-alanine mutant of Atg13 in atg13 -/-DT40 B cells did not block autophagy induction upon starvation [33].…”

Section: The Ulk1-atg13-fip200 Protein Kinase Complex and Protein Phomentioning

confidence: 99%

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Regulation of Autophagy by Protein Phosphorylation

Stork¹,

Alers²,

Löffler³

et al. 2012

Protein Phosphorylation in Human Health

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The role of the HORMA domain proteins ATG13 and ATG101 in initiating autophagosome biogenesis

Nguyen,

Faesen

2023

FEBS Letters

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Autophagy is a process of regulated degradation. It eliminates damaged and unnecessary cellular components by engulfing them with a de novo‐generated organelle: the double‐membrane autophagosome. The last three decades have provided us with a detailed parts list of the autophagy initiation machinery, have developed important insights into how these processes function and have identified regulatory proteins. It is now clear that autophagosome biogenesis requires the timely assembly of a complex machinery. However, it is unclear how a putative stable machine is assembled and disassembled, and how the different parts cooperate to perform its overall function. Although they have long been somewhat enigmatic in their precise role, HORMA domain proteins (first identified in Hop1p, Rev7p and MAD2 proteins) autophagy‐related protein 13 (ATG13) and ATG101 of the ULK‐kinase complex have emerged as important coordinators of the autophagy‐initiating subcomplexes. Here, we will particularly focus on ATG13 and ATG101 and the role of their unusual metamorphosis in initiating autophagosome biogenesis. We will also explore how this metamorphosis could potentially be purposefully rate‐limiting and speculate on how it could regulate the spontaneous self‐assembly of the autophagy‐initiating machinery.

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Atg13 and FIP200 act independently of Ulk1 and Ulk2 in autophagy induction

Cited by 119 publications

References 39 publications

Guidelines for the use and interpretation of assays for monitoring autophagy

Guidelines for the use and interpretation of assays for monitoring autophagy

Regulation of Autophagy by Protein Phosphorylation

The role of the HORMA domain proteins ATG13 and ATG101 in initiating autophagosome biogenesis

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