| 1. |
- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Forskningsöversikt (refereegranskat)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.
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| 2. |
- Knævelsrud, Helene, et al.
(författare)
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Membrane remodeling by the PX-BAR protein SNX18 promotes autophagosome formation
- 2013
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Ingår i: Journal of Cell Biology. - : Rockefeller University Press. - 0021-9525 .- 1540-8140. ; 202:2, s. 331-349
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Tidskriftsartikel (refereegranskat)abstract
- The membrane remodeling events required for autophagosome biogenesis are still poorly understood. Because PX domain proteins mediate membrane remodeling and trafficking, we conducted an imaging-based siRNA screen for autophagosome formation targeting human PX proteins. The PX-BAR protein SNX18 was identified as a positive regulator of autophagosome formation, and its Drosophila melanogaster homologue SH3PX1 was found to be required for efficient autophagosome formation in the larval fat body. We show that SNX18 is required for recruitment of Atg16L1-positive recycling endosomes to a perinuclear area and for delivery of Atg16L1- and LC3-positive membranes to autophagosome precursors. We identify a direct interaction of SNX18 with LC3 and show that the pro-autophagic activity of SNX18 depends on its membrane binding and tubulation capacity. We also show that the function of SNX18 in membrane tubulation and autophagy is negatively regulated by phosphorylation of S233. We conclude that SNX18 promotes autophagosome formation by virtue of its ability to remodel membranes and provide membrane to forming autophagosomes.
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| 3. |
- O'Farrell, Fergal, et al.
(författare)
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Two-Tiered Control of Epithelial Growth and Autophagy by the Insulin Receptor and the Ret-Like Receptor, Stitcher
- 2013
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Ingår i: PLoS biology. - : Public Library of Science (PLoS). - 1544-9173 .- 1545-7885. ; 11:7, s. e1001612-
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Tidskriftsartikel (refereegranskat)abstract
- Body size in Drosophila larvae, like in other animals, is controlled by nutrition. Nutrient restriction leads to catabolic responses in the majority of tissues, but the Drosophila mitotic imaginal discs continue growing. The nature of these differential control mechanisms that spare distinct tissues from starvation are poorly understood. Here, we reveal that the Ret-like receptor tyrosine kinase (RTK), Stitcher (Stit), is required for cell growth and proliferation through the PI3K-I/TORC1 pathway in the Drosophila wing disc. Both Stit and insulin receptor (InR) signaling activate PI3K-I and drive cellular proliferation and tissue growth. However, whereas optimal growth requires signaling from both InR and Stit, catabolic changes manifested by autophagy only occur when both signaling pathways are compromised. The combined activities of Stit and InR in ectodermal epithelial tissues provide an RTK-mediated, two-tiered reaction threshold to varying nutritional conditions that promote epithelial organ growth even at low levels of InR signaling.
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| 4. |
- Schultz, Sebastian, et al.
(författare)
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HIAPP and hproIAPP triggers elective autophagy and inhibit the neuro-protective effect of autophagy
- 2010
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Introduction: Amyloid formation is associated with cell death and islet amyloid is thought to participate in the 50-60% β-cell reduction detected in patients with type 2 diabetes. Islet amyloid polypeptide (IAPP) is the main amyloid protein in the islets of Langerhans. Initial IAPP-amyloid formation is intracellular and part of this amyloid constitutes of proIAPP. Material & methods: We have established a new model in Drosophila melanogaster where expression of hproIAPP and IAPP results in the formation of amyloid. With this model, we have investigated the effect of protein aggregation on pathways such as ER-stress, unfolded protein response (UPR), apoptosis and autophagy. Important steps in the different pathways were manipulated by RNAi-technique or over- expression of endogenous Drosophila proteins. Results: Expression of hproIAPP and hIAPP driven to the pdf-neurons led to cell death, but this was without activation of ER-stress, UPR or apoptosis. Aggregated hproIAPP and IAPP, labeled with antibodies against ubiquitin and p62 were accumulated intracellular, a finding that points to an involvement of autophagy. HproIAPP and hIAPP were shown to exert their toxic activity by an intracellular mechanism in contrary to Aβ42 and Aβ42 E22G that exhibit an extracellular toxic activity. Conclusion: Studies on toxicity suggest that hproIAPP and hIAPP aggregates can occupy the autophagy pathway and prevent maintenance of basal cellular homeostasis. Comparison of proIAPP/IAPP and Aβ42 toxicity shows that amyloid proteins of separate origin can exhibit different toxicity.
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