Iwona A. Ciechomska

Bio: Iwona A. Ciechomska is an academic researcher from Nencki Institute of Experimental Biology. The author has contributed to research in topics: Autophagy & Glioma. The author has an hindex of 18, co-authored 29 publications receiving 5868 citations. Previous affiliations of Iwona A. Ciechomska include University of Cambridge.

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

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. For example, 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 versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase 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. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. 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. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. 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 assays, we hope to encourage technical innovation in the field.

Bcl-2 complexed with Beclin-1 maintains full anti-apoptotic function.

Abstract: The binding of Bcl-2 to Beclin-1 reduces Beclin-1's capacity to induce autophagy. Here, we have tested whether the interaction is reciprocated by loss of Bcl-2's anti-apoptotic function. We targeted Bcl-2 to mitochondria or endoplasmic reticulum (ER) and induced apoptosis using several apoptotic stimuli that initiate ER and/or mitochondrial signaling pathways (UV radiation, TNF and cycloheximide, staurosporine, thapsigargin and tunicamycin). When Beclin-1 and Bcl-2 were expressed together in HeLa cells, Beclin-1 (but not Beclin-1 lacking the Bcl-2-binding domain) followed Bcl-2 to the appropriate organelle with complete or near-complete overlap (comprising 60 and 30% of cells, respectively). The interaction between Beclin-1 and Bcl-2 was verified by immunoprecipitation, and a membrane-proximate localization of Beclin-1 was shown by immunoelectron microscopy. Apoptosis was followed by measuring changes in nuclear morphology, caspase-3 activity, poly-ADP-ribose polymerase cleavage or punctation of mRFP-Bax on mitochondria. Binding of Beclin-1 to Bcl-2 did not modify apoptosis irrespective of Bcl-2 concentration, location or apoptotic stimulus. A similar result was obtained in Atg5-/- cells that are autophagy-deficient, arguing against compensation for the loss of protection by Bcl-2 by autophagy-mediated survival induced by Beclin-1. Hence, although Beclin-1 contains a BH3-only motif typical of pro-apoptotic proteins, it is a negligible modulator of Bcl-2's anti-apoptotic function.

Endoplasmic reticulum stress triggers autophagy in malignant glioma cells undergoing cyclosporine a-induced cell death.

Abstract: Autophagy is a conserved, self-digestion process that is activated in response to nutrient limitation but acting also as an alternative death mechanism under certain conditions. It is accompanied by the progressive formation of vesicle structures from autophagosomes to autophagolysosomes orchestrated by autophagy effectors (Atg proteins) and modulators (that is, mTOR-mammalian target of rapamycin as a negative regulator). Malignant gliomas are highly resistant to current therapies that induce apoptosis, thus induction of the alternative cell death is an attractive strategy. We demonstrate that cyclosporine A (CsA, an immunophilin/calcineurin inhibitor) induces cell death with some apoptotic features but also accompanied by the appearance of numerous cytoplasmic vacuoles, immunostained for endoplasmic reticulum (ER) and autophagy markers. The induction of ER stress in glioma cells by CsA was evidenced by detection of unfolded protein response activation (phosphorylation of PERK, accumulation of IRE1α) and accumulation of ER stress-associated proteins (BIP and CHOP). Formation of the acidic vesicular organelles, increase of autophagic vacuoles, GFP-LC3 punctation (microtubule-associated protein light chain 3) and LC3-II accumulation upon CsA treatment confirmed activation of autophagy. Decrease of phosphorylation of 4E-BP1, p70S6K1 and its downstream target S6 ribosomal protein demonstrate inhibition of mTOR signaling by CsA. Salubrinal and silencing of PERK and IRE1α partially blocked CsA-induced accumulation of LC3-II. It suggests that ER stress precedes CsA-induced autophagy. Surprisingly, silencing of autophagy effectors ULK1, Atg5 or Atg7 increased the level of active caspases 3, 7 and PARP degradation in CsA-treated cells. Our results demonstrate that CsA induces both apoptosis and autophagy in malignant glioma cells via induction of ER stress and inhibition of mTOR/p70S6K1 pathway, however autophagy is cytoprotective in this context.

Inhibition of Akt kinase signalling and activation of Forkhead are indispensable for upregulation of FasL expression in apoptosis of glioma cells

Abstract: Activation of Akt signalling pathway is frequently found in glioma cells and may contribute to their resistance to undergo apoptosis in response to conventional therapies. We found that cyclosporin A (CsA) induces apoptosis of C6 glioma cells, which is associated with transcriptional activation of fasL. In the present paper, we investigated an involvement of Akt signalling in the regulation of FasL expression in CsA-induced apoptosis. We demonstrated that the level of active Akt decreases significantly after CsA treatment, which results in the decrease of Forkhead phosphorylation and its translocation to the nucleus. It correlated with an increase of binding to the Forkhead-responsive element FHRE from the FasL promoter, as demonstrated by gel-shift assays. Although treatment with LY294002, a specific inhibitor of PI3 K, decreased the phosphorylation of Akt and increased Fkhr translocation to the nucleus, these events were not sufficient to induce FasL expression and apoptosis of C6 glioma cells. Interference with Akt/Forkhead signalling by membrane-targeted Akt or removal of the FKHR-binding sites from the FasL promoter significantly abolished its activation. These results indicate that downregulation of Akt signalling and activation of Forkhead is a prerequisite for the induction of FasL promoter. It may be clinically important for pharmacological intervention in gliomas.

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

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. For example, 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 versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase 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. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. 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. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. 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 assays, we hope to encourage technical innovation in the field.

Guidelines for the use and interpretation of assays for monitoring autophagy

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.

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Abstract: Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

Mammalian caspases: structure ,a ctivation ,s ubstrates, and functions during apoptosis

Abstract: ■ Abstract Apoptosis is a genetically programmed, morphologically distinct form of cell death that can be triggered by a variety of physiological and pathological stimuli. Studies performed over the past 10 years have demonstrated that proteases play critical roles in initiation and execution of this process. The caspases, a family of cysteine-dependent aspartate-directed proteases, are prominent among the death proteases. Caspases are synthesized as relatively inactive zymogens that become activated by scaffold-mediated transactivation or by cleavage via upstream proteases in an intracellular cascade. Regulation of caspase activation and activity occurs at several different levels: ( a) Zymogen gene transcription is regulated; ( b) antiapoptotic members of the Bcl-2 family and other cellular polypeptides block proximity-induced activation of certain procaspases; and ( c) certain cellular inhibitor of apoptosis proteins (cIAPs) can bind to and inhibit active caspases. Once activated, caspases cleave a variety of intracellular polypeptides, including major structural elements of the cytoplasm and nucleus, components of the DNA repair machinery, and a number of protein kinases. Collectively, these scissions disrupt survival pathways and disassemble important architectural components of the cell, contributing to the stereotypic morphological and biochemical changes that characterize apoptotic cell death.

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

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