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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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- Galluzzi, L, et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes.
- 2009
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Ingår i: Cell death and differentiation. - : Springer Science and Business Media LLC. - 1476-5403 .- 1350-9047. ; 16:8, s. 1093-107
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Forskningsöversikt (refereegranskat)abstract
- Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.
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- Levi-Montalcini, R, et al.
(författare)
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Rita's 102!!
- 2011
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Ingår i: Molecular neurobiology. - 1559-1182. ; 43:2, s. 77-79
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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- Green, DR, et al.
(författare)
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Ten years of publication in cell death
- 2004
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Ingår i: Cell death and differentiation. - : Springer Science and Business Media LLC. - 1350-9047 .- 1476-5403. ; 11:1, s. 2-3
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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- Melino, G, et al.
(författare)
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On Rita Levi-Montalcini
- 2013
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Ingår i: Molecular neurobiology. - 1559-1182. ; 47:2, s. 443-445
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Tidskriftsartikel (refereegranskat)
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- Wilhelm, MT, et al.
(författare)
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Isoform-specific p73 knockout mice reveal a novel role for delta Np73 in the DNA damage response pathway
- 2010
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Ingår i: Genes & development. - : Cold Spring Harbor Laboratory. - 1549-5477 .- 0890-9369. ; 24:6, s. 549-560
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Tidskriftsartikel (refereegranskat)abstract
- Mice with a complete deficiency of p73 have severe neurological and immunological defects due to the absence of all TAp73 and ΔNp73 isoforms. As part of our ongoing program to distinguish the biological functions of these isoforms, we generated mice that are selectively deficient for the ΔNp73 isoform. Mice lacking ΔNp73 (ΔNp73−/− mice) are viable and fertile but display signs of neurodegeneration. Cells from ΔNp73−/− mice are sensitized to DNA-damaging agents and show an increase in p53-dependent apoptosis. When analyzing the DNA damage response (DDR) in ΔNp73−/− cells, we discovered a completely new role for ΔNp73 in inhibiting the molecular signal emanating from a DNA break to the DDR pathway. We found that ΔNp73 localizes directly to the site of DNA damage, can interact with the DNA damage sensor protein 53BP1, and inhibits ATM activation and subsequent p53 phosphorylation. This novel finding may explain why human tumors with high levels of ΔNp73 expression show enhanced resistance to chemotherapy.
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