strategies-for-tracking-anastasis-cell-survival-phenomenon-that

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

in-vivo-biosensor-tracks-non-apoptotic-caspase-activity-in-drosophila

In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila

Caspases are the key mediators of apoptotic cell death via their proteolytic activity. When caspases are activated in cells to levels detectable by ...

detecting-anastasis-in-vivo-by-caspasetracker-biosensor

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death ...

Vimentin is one of the intermediate filaments that functions in structural support, signal transduction and organelle positioning of a cell. In the present study, we report the contribution of vimentin in mitochondrial morphology and organization. Using subcellular fractionation, immunoprecipitation and fluorescence microscopy analyses, we found that vimentin was associated with mitochondria. Knockdown of vimentin resulted in mitochondrial fragmentation, swelling and disorganization. We further demonstrated that the vimentin cytoskeleton co-localized and interacted with mitochondria to a greater extent than other cytoskeletal components known to support mitochondria. Our results also suggest that vimentin could participate in the mitochondrial association of microtubules. As mitochondrial morphologies determine mitochondrial function, our findings revealed a potentially important relationship between the vimentin-based intermediate filaments and the regulation of mitochondria.

Vimentin supports mitochondrial morphology and organization.

The Mu transposon system of maize is highly active, with each of the approximately 50-100 copies transposing on average once each generation. The approximately one dozen distinct Mu transposons contain highly similar approximately 215 bp terminal inverted repeats (TIRs) and generate 9-bp target site duplications (TSDs) upon insertion. Using a novel genome walking strategy that uses these conserved TIRs as primer binding sites, Mu insertion sites were amplified from Mu stocks and sequenced via 454 technology. 94% of approximately 965,000 reads carried Mu TIRs, demonstrating the specificity of this strategy. Among these TIRs, 21 novel Mu TIRs were discovered, revealing additional complexity of the Mu transposon system. The distribution of >40,000 non-redundant Mu insertion sites was strikingly non-uniform, such that rates increased in proportion to distance from the centromere. An identified putative Mu transposase binding consensus site does not explain this non-uniformity. An integrated genetic map containing more than 10,000 genetic markers was constructed and aligned to the sequence of the maize reference genome. Recombination rates (cM/Mb) are also strikingly non-uniform, with rates increasing in proportion to distance from the centromere. Mu insertion site frequencies are strongly correlated with recombination rates. Gene density does not fully explain the chromosomal distribution of Mu insertion and recombination sites, because pronounced preferences for the distal portion of chromosome are still observed even after accounting for gene density. The similarity of the distributions of Mu insertions and meiotic recombination sites suggests that common features, such as chromatin structure, are involved in site selection for both Mu insertion and meiotic recombination. The finding that Mu insertions and meiotic recombination sites both concentrate in genomic regions marked with epigenetic marks of open chromatin provides support for the hypothesis that open chromatin enhances rates of both Mu insertion and meiotic recombination.

Mu transposon insertion sites and meiotic recombination events co-localize with epigenetic marks for open chromatin across the maize genome.

Apoptosis serves as a protective mechanism by eliminating damaged cells through programmed cell death. After apoptotic cells pass critical checkpoints, including mitochondrial fragmentation, executioner caspase activation, and DNA damage, it is assumed that cell death inevitably follows. However, this assumption has not been tested directly. Here we report an unexpected reversal of late-stage apoptosis in primary liver and heart cells, macrophages, NIH 3T3 fibroblasts, cervical cancer HeLa cells, and brain cells. After exposure to an inducer of apoptosis, cells exhibited multiple morphological and biochemical hallmarks of late-stage apoptosis, including mitochondrial fragmentation, caspase-3 activation, and DNA damage. Surprisingly, the vast majority of dying cells arrested the apoptotic process and recovered when the inducer was washed away. Of importance, some cells acquired permanent genetic changes and underwent oncogenic transformation at a higher frequency than controls. Global gene expression analysis identified a molecular signature of the reversal process. We propose that reversal of apoptosis is an unanticipated mechanism to rescue cells from crisis and propose to name this mechanism "anastasis" (Greek for "rising to life"). Whereas carcinogenesis represents a harmful side effect, potential benefits of anastasis could include preservation of cells that are difficult to replace and stress-induced genetic diversity.

Cell survival, DNA damage, and oncogenic transformation after a transient and reversible apoptotic response.

Apoptosis dient als Schutzmechanismus durch Eliminierung defekter Zellen durch programmierten Zelltod. Nachdem Apoptotic Zellen kritische Kontrollpunkte, mitochondriale Fragmentierung Scharfrichter Caspase Aktivierung und DNA-Sch&auml;den, &uuml;bergeben ist davon auszugehen, dass Zellentod unweigerlich folgt. Diese Annahme wurde jedoch nicht direkt getestet. Hier berichten wir eine unerwartete Umlenkung des Sp&auml;tstadiums Apoptosis in den prim&auml;ren Leber und Herz-Zellen, Makrophagen, NIH 3T3 Fibroblasten, Geb&auml;rmutterhalskrebs HeLa-Zellen und Gehirnzellen. Nach Exposition gegen&uuml;ber einem Induktor von Apoptosis ausgestellt Zellen mehrere morphologische und Biochemische Merkmale des Sp&auml;tstadiums Apoptosis, mitochondriale Fragmentierung Caspase-3-Aktivierung und DNA-Sch&auml;den. &Uuml;berraschend, dass die &uuml;berwiegende Mehrheit der sterbende Zellen verhaftet den apoptotischen Prozess und wiederhergestellt, wenn der Induktor weggesp&uuml;lt wurde. Wichtig einige Zellen erworben dauerhafte genetische Ver&auml;nderungen und unterzog sich Onkogene Transformation bei einer h&ouml;heren Frequenz als Steuerelemente. Globale Genexpressionsanalyse identifiziert eine molekulare Signatur des Umkehr-Prozesses. Wir schlagen vor, die Umkehrung der Apoptose ist eine unerwartete Mechanismus, um Zellen aus der Krise zu retten und vorschlagen, diesen Mechanismus &quot;Anastasis&quot; (griechisch f&uuml;r &quot;Rising zum Leben&quot;) zu nennen. W&auml;hrend Karzinogenese eine sch&auml;dliche Nebenwirkung darstellt, k&ouml;nnten potenzielle Anastasis Vorteile Erhaltung der Zellen, die nur schwer zu ersetzen sind und Stress-induced genetische Vielfalt.

Zelle &uuml;berleben, DNA-Sch&auml;den und Onkogene Transformation nach eine vor&uuml;bergehende und reversible Apoptotic Antwort.

Bulked segregant analysis (BSA) is an efficient method to rapidly and efficiently map genes responsible for mutant phenotypes. BSA requires access to quantitative genetic markers that are polymorphic in the mapping population. We have developed a modification of BSA (BSR-Seq) that makes use of RNA-Seq reads to efficiently map genes even in populations for which no polymorphic markers have been previously identified. Because of the digital nature of next-generation sequencing (NGS) data, it is possible to conduct de novo SNP discovery and quantitatively genotype BSA samples by analyzing the same RNA-Seq data using an empirical Bayesian approach. In addition, analysis of the RNA-Seq data provides information on the effects of the mutant on global patterns of gene expression at no extra cost. In combination these results greatly simplify gene cloning experiments. To demonstrate the utility of this strategy BSR-Seq was used to clone the glossy3 (gl3) gene of maize. Mutants of the glossy loci exhibit altered accumulation of epicuticular waxes on juvenile leaves. By subjecting the reference allele of gl3 to BSR-Seq, we were able to map the gl3 locus to an ≈ 2 Mb interval. The single gene located in the ≈ 2 Mb mapping interval whose expression was down-regulated in the mutant pool was subsequently demonstrated to be the gl3 gene via the analysis of multiple independent transposon induced mutant alleles. The gl3 gene encodes a putative myb transcription factor, which directly or indirectly affects the expression of a number of genes involved in the biosynthesis of very-long-chain fatty acids.

Gene mapping via bulked segregant RNA-Seq (BSR-Seq).

The midribs of maize brown midrib (bm) mutants exhibit a reddish-brown color associated with reductions in lignin concentration and alterations in lignin composition. Here, we report the mapping, cloning, and functional and biochemical analyses of the bm2 gene. The bm2 gene was mapped to a small region of chromosome 1 that contains a putative methylenetetrahydrofolate reductase (MTHFR) gene, which is down-regulated in bm2 mutant plants. Analyses of multiple Mu-induced bm2-Mu mutant alleles confirmed that this constitutively expressed gene is bm2. Yeast complementation experiments and a previously published biochemical characterization show that the bm2 gene encodes a functional MTHFR. Quantitative RT-PCR analyses demonstrated that the bm2 mutants accumulate substantially reduced levels of bm2 transcript. Alteration of MTHFR function is expected to influence accumulation of the methyl donor S-adenosyl-L-methionine (SAM). Because SAM is consumed by two methyltransferases in the lignin pathway (Ye et al., ), the finding that bm2 encodes a functional MTHFR is consistent with its lignin phenotype. Consistent with this functional assignment of bm2, the expression patterns of genes in a variety of SAM-dependent or -related pathways, including lignin biosynthesis, are altered in the bm2 mutant. Biochemical assays confirmed that bm2 mutants accumulate reduced levels of lignin with altered composition compared to wild-type. Hence, this study demonstrates a role for MTHFR in lignin biosynthesis.

The maize brown midrib2 (bm2) gene encodes a methylenetetrahydrofolate reductase that contributes to lignin accumulation.

Root hairs are instrumental for nutrient uptake in monocot cereals. The maize (Zea mays L.) roothairless5 (rth5) mutant displays defects in root hair initiation and elongation manifested by a reduced density and length of root hairs. Map-based cloning revealed that the rth5 gene encodes a monocot-specific NADPH oxidase. RNA-Seq, in situ hybridization and qRT-PCR experiments demonstrated that the rth5 gene displays preferential expression in root hairs but also accumulates to low levels in other tissues. Immunolocalization detected RTH5 proteins in the epidermis of the elongation and differentiation zone of primary roots. Because superoxide and hydrogen peroxide levels are reduced in the tips of growing rth5 mutant root hairs as compared with wild-type, and Reactive oxygen species (ROS) is known to be involved in tip growth, we hypothesize that the RTH5 protein is responsible for establishing the high levels of ROS in the tips of growing root hairs required for elongation. Consistent with this hypothesis, a comparative RNA-Seq analysis of 6-day-old rth5 versus wild-type primary roots revealed significant over-representation of only two gene ontology (GO) classes related to the biological functions (i.e. oxidation/reduction and carbohydrate metabolism) among 893 differentially expressed genes (FDR <5%). Within these two classes the subgroups 'response to oxidative stress' and 'cellulose biosynthesis' were most prominently represented.

Roothairless5, which functions in maize (Zea mays L.) root hair initiation and elongation encodes a monocot-specific NADPH oxidase.

Mutations in the brown midrib4 (bm4) gene affect the accumulation and composition of lignin in maize. Fine-mapping analysis of bm4 narrowed the candidate region to an approximately 105 kb interval on chromosome 9 containing six genes. Only one of these six genes, GRMZM2G393334, showed decreased expression in mutants. At least four of 10 Mu-induced bm4 mutant alleles contain a Mu insertion in the GRMZM2G393334 gene. Based on these results, we concluded that GRMZM2G393334 is the bm4 gene. GRMZM2G393334 encodes a putative folylpolyglutamate synthase (FPGS), which functions in one-carbon (C1) metabolism to polyglutamylate substrates of folate-dependent enzymes. Yeast complementation experiments demonstrated that expression of the maize bm4 gene in FPGS-deficient met7 yeast is able to rescue the yeast mutant phenotype, thus demonstrating that bm4 encodes a functional FPGS. Consistent with earlier studies, bm4 mutants exhibit a modest decrease in lignin concentration and an overall increase in the S:G lignin ratio relative to wild-type. Orthologs of bm4 include at least one paralogous gene in maize and various homologs in other grasses and dicots. Discovery of the gene underlying the bm4 maize phenotype illustrates a role for FPGS in lignin biosynthesis.

The maize brown midrib4 (bm4) gene encodes a functional folylpolyglutamate synthase.

The discovery that mammalian cells can survive late-stage apoptosis challenges the general assumption that active caspases are markers of impending death. However, tools have not been available to track healthy cells that have experienced caspase activity at any time in the past. Therefore, to determine if cells in whole animals can undergo reversal of apoptosis, known as anastasis, we developed a dual color CaspaseTracker system for Drosophila to identify cells with ongoing or past caspase activity. Transient exposure of healthy females to environmental stresses such as cold shock or starvation activated the CaspaseTracker coincident with caspase activity and apoptotic morphologies in multiple cell types of developing egg chambers. Importantly, when stressed flies were returned to normal conditions, morphologically healthy egg chambers and new progeny flies were labeled by the biosensor, suggesting functional recovery from apoptotic caspase activation. In striking contrast to developing egg chambers, which lack basal caspase biosensor activation under normal conditions, many adult tissues of normal healthy flies exhibit robust caspase biosensor activity in a portion of cells, including neurons. The widespread persistence of CaspaseTracker-positivity implies that healthy cells utilize active caspases for non-apoptotic physiological functions during and after normal development.

In vivo CaspaseTracker biosensor system for detecting anastasis and non-apoptotic caspase activity.

Anastasis (Greek for "rising to life") is a cell recovery phenomenon that rescues dying cells from the brink of cell death. We recently discovered anastasis to occur after the execution-stage of apoptosis in vitro and in vivo. Promoting anastasis could in principle preserve injured cells that are difficult to replace, such as cardiomyocytes and neurons. Conversely, arresting anastasis in dying cancer cells after cancer therapies could improve treatment efficacy. To develop new therapies that promote or inhibit anastasis, it is essential to identify the key regulators and mediators of anastasis - the therapeutic targets. Therefore, we performed time-course microarray analysis to explore the molecular mechanisms of anastasis during reversal of ethanol-induced apoptosis in mouse primary liver cells. We found striking changes in transcription of genes involved in multiple pathways, including early activation of pro-cell survival, anti-oxidation, cell cycle arrest, histone modification, DNA-damage and stress-inducible responses, and at delayed times, angiogenesis and cell migration. Validation with RT-PCR confirmed similar changes in the human liver cancer cell line, HepG2, during anastasis. Here, we present the time-course whole-genome gene expression dataset revealing gene expression profiles during the reversal of apoptosis. This dataset provides important insights into the physiological, pathological, and therapeutic implications of anastasis.

Molecular signature of anastasis for reversal of apoptosis.

Mannose receptor (MRC1/CD206) has been suggested to mediate allergic sensitization and asthma to multiple glycoallergens, including cockroach allergens.

Mannose receptor modulates macrophage polarization and allergic inflammation through miR-511-3p.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is associated with mast cell-mediated inflammation and heightened oxidant stress. Kynurenine (KYN), an endogenous tryptophan metabolite, can promote allergen-induced mast cell activation through the aryl hydrocarbon receptor (AhR).

ArticleTotal : 3	Year
Publication title Cited by 32	2015
Publication title Cited by 5	2016
Publication title Cited by 17	2018

Article	Year
Vimentin supports mitochondrial morphology and organization. The Biochemical journal\| PubMed ID: 17983357	2008
Mu transposon insertion sites and meiotic recombination events co-localize with epigenetic marks for open chromatin across the maize genome. PLoS genetics\| PubMed ID: 19936291	2009
Cell survival, DNA damage, and oncogenic transformation after a transient and reversible apoptotic response. Molecular biology of the cell\| PubMed ID: 22535522	2012
Gene mapping via bulked segregant RNA-Seq (BSR-Seq). PloS one\| PubMed ID: 22586469	2012
The maize brown midrib2 (bm2) gene encodes a methylenetetrahydrofolate reductase that contributes to lignin accumulation. The Plant journal : for cell and molecular biology\| PubMed ID: 24286468	2014
Roothairless5, which functions in maize (Zea mays L.) root hair initiation and elongation encodes a monocot-specific NADPH oxidase. The Plant journal : for cell and molecular biology\| PubMed ID: 24902980	2014
The maize brown midrib4 (bm4) gene encodes a functional folylpolyglutamate synthase. The Plant journal : for cell and molecular biology\| PubMed ID: 25495051	2015
In vivo CaspaseTracker biosensor system for detecting anastasis and non-apoptotic caspase activity. Scientific reports\| PubMed ID: 25757939	2015
Molecular signature of anastasis for reversal of apoptosis. F1000Research\| PubMed ID: 28299189	2017
Mannose receptor modulates macrophage polarization and allergic inflammation through miR-511-3p. The Journal of allergy and clinical immunology\| PubMed ID: 28629744	2018
Functional role of kynurenine and aryl hydrocarbon receptor axis in chronic rhinosinusitis with nasal polyps. The Journal of allergy and clinical immunology\| PubMed ID: 28689792	2017

Ho Man Tang

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