| 1. |
- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes
- 2008
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Ingår i: Autophagy. - : Landes Bioscience. - 1554-8627 .- 1554-8635. ; 4:2, s. 151-175
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Forskningsöversikt (refereegranskat)abstract
- Research in autophagy continues to accelerate,1 and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.2,3 There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is 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 verify an autophagic response.
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| 2. |
- Kurz, Tino, et al.
(författare)
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Lysosomes In Iron Metabolism, Ageing And Apoptosis
- 2008
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Ingår i: Histochemistry and Cell Biology. - : Institutionen för medicin och hälsa. - 0948-6143 .- 1432-119X. ; 129:4, s. 389-406
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Tidskriftsartikel (refereegranskat)abstract
- The lysosomal compartment is essential for a variety of cellular functions, including the normal turnover of most long-lived proteins and all organelles. The compartment consists of numerous acidic vesicles (pH ~4-5) that constantly fuse and divide. It receives a large number of hydrolases (~50) from the trans-Golgi network, and substrates from both the cells’ outside (heterophagy) and inside (autophagy). Many macromolecules contain iron that gives rise to an iron-rich environment in lysosomes that recently have degraded such macromolecules. Iron-rich lysosomes are sensitive to oxidative stress, while ‘resting’ lysosomes, which have not recently participated in autophagic events, are not. The magnitude of oxidative stress determines the degree of lysosomal destabilization and, consequently, whether arrested growth, reparative autophagy, apoptosis, or necrosis will follow. Heterophagy is the first step in the process by which immunocompetent cells modify antigens and produce antibodies, while exocytosis of lysosomal enzymes may promote tumor invasion, angiogenesis, and metastasis. Apart from being an essential turnover process, autophagy is also a mechanism by which cells will be able to sustain temporary starvation and rid themselves of intracellular organisms that have invaded, although some pathogens have evolved mechanisms to prevent their destruction. Mutated lysosomal enzymes are the underlying cause of a number of lysosomal storage diseases involving the accumulation of materials that would be the substrate for the corresponding hydrolases, were they not defective. The normal, low-level diffusion of hydrogen peroxide into iron-rich lysosomes causes the slow formation of lipofuscin in long-lived postmitotic cells, where it occupies a substantial part of the lysosomal compartment at the end of the life span. This seems to result in the diversion of newly produced lysosomal enzymes away from autophagosomes, leading to the accumulation of malfunctioning mitochondria and proteins with consequent cellular dysfunction. If autophagy were a perfect turnover process, postmitotic ageing and several age-related neurodegenerative diseases would, perhaps, not take place.
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| 3. |
- Stroikin, Yuri, et al.
(författare)
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Testing the “garbage” accumulation theory of ageing : mitotic activity protects cells from death induced by inhibition of autophagy
- 2005
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Ingår i: Biogerontology. - : Springer Science and Business Media LLC. - 1389-5729 .- 1573-6768. ; 6:1, s. 39-47
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Tidskriftsartikel (refereegranskat)abstract
- Imperfect autophagic degradation of oxidatively damaged macromolecules and organelles (so-called biological garbage) is considered an important contributor to ageing and consequent death of postmitotic (non-dividing) cells, such as neurons and cardiac myocytes. In contrast, proliferating cells apparently escape senescence by a continuous dilution and repair of damaged structures during division. Postmitotic ageing can be mimicked and studied in cultures of potentially dividing cells if their mitotic activity is inhibited. To test the garbage accumulation theory of ageing, we compared survival of density-dependent growth-arrested (confluent) and proliferating human fibroblasts and astrocytes following inhibition of autophagic sequestration with 3-methyladenine (3MA). Exposure of confluent fibroblast cultures to 3MA for two weeks resulted in a significantly increased proportion of dying cells compared to both untreated confluent cultures and dividing cells with 3MA-inhibited autophagy. Similar results were obtained when autophagic degradation was suppressed by the protease inhibitor leupeptin. In 3MA- or leupeptin-exposed cultures, dying cells were overloaded with undegraded autofluorescent material. The results support a key role of biological lysosomal garbage accumulation in the triggering of ageing and death of postmitotic cells, as well as the anti-ageing role of cell division.
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