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- 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. |
- Chen, Mei-Qin, et al.
(författare)
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Arabidopsis NMD3 is required for nuclear export of 60S ribosomal subunits and affects secondary cell wall thickening
- 2012
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:4, s. 35904-35904
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Tidskriftsartikel (refereegranskat)abstract
- NMD3 is required for nuclear export of the 60S ribosomal subunit in yeast and vertebrate cells, but no corresponding function of NMD3 has been reported in plants. Here we report that Arabidopsis thaliana NMD3 (AtNMD3) showed a similar function in the nuclear export of the 60S ribosomal subunit. Interference with AtNMD3 function by overexpressing a truncated dominant negative form of the protein lacking the nuclear export signal sequence caused retainment of the 60S ribosomal subunits in the nuclei. More interestingly, the transgenic Arabidopsis with dominant negative interference of AtNMD3 function showed a striking failure of secondary cell wall thickening, consistent with the altered expression of related genes and composition of cell wall components. Observation of a significant decrease of rough endoplasmic reticulum (RER) in the differentiating interfascicular fiber cells of the transgenic plant stems suggested a link between the defective nuclear export of 60S ribosomal subunits and the abnormal formation of the secondary cell wall. These findings not only clarified the evolutionary conservation of NMD3 functions in the nuclear export of 60S ribosomal subunits in yeast, animals and plants, but also revealed a new facet of the regulatory mechanism underlying secondary cell wall thickening in Arabidopsis. This new facet is that the nuclear export of 60S ribosomal subunits and the formation of RER may play regulatory roles in coordinating protein synthesis in cytoplasm and transcription in nuclei.
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| 3. |
- Wei, Tao, et al.
(författare)
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Molecular and catalytic characterization of a phi class glutathione transferase from Cathaya argyrophylla
- 2012
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Ingår i: Biochemical Systematics and Ecology. - Oxford : Pergamon Press. - 0305-1978 .- 1873-2925. ; 40, s. 75-85
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Tidskriftsartikel (refereegranskat)abstract
- Plant phi class glutathione transferases (GSTs) play important roles in stress tolerance and detoxification metabolism. This study reports the cloning, expression and biochemical characteristics of a phi GST gene (CaGSTF) from the endemic and endangered conifer Cathaya argyrophylla. The recombinant CaGSTF showed GSH-conjugating activity towards the substrate NED-Cl and CDNB. Kinetic analysis revealed low catalytic efficiency with a k(cat)/K-m(GSH) value of 9.82 mM(-1)S(-1). The CaGSTF proved to be a thermolabile enzyme, at 40 degrees C the enzyme's activity was nearly abolished. Site-directed mutagenesis revealed that Ser12, Lys42, Ile55, Glu67 and Ser68 of CaGSTF are critical components of glutathione-binding sites that contribute to the enzyme's catalytic activity. Compared to other plant phi GSTs and conifer tau GSTs, CaGSTF showed a narrow substrate spectrum, low catalytic efficiency and thermolability. These atypical properties suggest the enzyme may have a limited functional role in the organism's adaptation to environmental stresses in the subtropical regions. (C) 2011 Elsevier Ltd. All rights reserved.
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