scholarly journals Live Imaging of muscle histolysis inDrosophilametamorphosis

2016 ◽  
Author(s):  
Yadav Kuleesha ◽  
Wee Choo Puah ◽  
Martin Wasser
Keyword(s):  
Cell Death ◽  
Molecular Mechanisms ◽  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Time Lapse ◽  
Live Imaging ◽  
Dominant Negative ◽  
Muscle Size ◽  
Genes Encoding

Background:The contribution of programmed cell death (PCD) to muscle wasting disorders remains a matter of debate.Drosophila melanogastermetamorphosis offers the opportunity to study muscle cell death in the context of development. Using live cell imaging of the abdomen, two groups of larval muscles can be observed, doomed muscles that undergo histolysis and persistent muscles that are remodelled and survive into adulthood.Method:To identify and characterize genes that control the decision between survival and cell death of muscles, we developed a method comprisingin vivoimaging, targeted gene perturbation and time-lapse image analysis. Our approach enabled us to study the cytological and temporal aspects of abnormal cell death phenotypes.Results:In a previous genetic screen for genes controlling muscle size and cell death in metamorphosis, we identified gene perturbations that induced cell death of persistent or inhibit histolysis of doomed larval muscles. RNA interference (RNAi) of the genes encoding the helicase Rm62 and the lysosomal Cathepsin-L homolog Cysteine proteinase 1 (Cp1) caused premature cell death of persistent muscle in early and mid-pupation, respectively. Silencing of the transcriptional co-repressorAtrophininhibited histolysis of doomed muscles. Overexpression of dominant-negative Target of Rapamycin (TOR) delayed the histolysis of a subset of doomed and induced ablation of all persistent muscles. RNAi ofAMPKα,which encodes a subunit of the AMPK protein complex that senses AMP and promotes ATP formation, led to loss of attachment and a spherical morphology. None of the perturbations affected the survival of newly formed adult muscles, suggesting that the method is useful to find genes that are crucial for the survival of metabolically challenged muscles, like those undergoing atrophy. The ablation of persistent muscles did not affect eclosion of adult flies.Conclusions:Live imaging is a versatile approach to uncover gene functions that are required for the survival of muscle undergoing remodelling, yet are dispensable for other adult muscles. Our approach promises to identify molecular mechanisms that can explain the resilience of muscles to PCD.

scholarly journals A unique mode of keratinocyte death requires intracellular acidification

2021 ◽  
Vol 118 (17) ◽  
pp. e2020722118
Author(s):  
Takeshi Matsui ◽  
Nanako Kadono-Maekubo ◽  
Yoshiro Suzuki ◽  
Yuki Furuichi ◽  
Keiichiro Shiraga ◽  
...  
Keyword(s):  
Cell Death ◽  
Molecular Mechanisms ◽  
Transient Receptor Potential ◽  
Nuclear Dna ◽  
Receptor Potential ◽  
Time Lapse ◽  
Intravital Imaging ◽  
Death Process ◽  
Intracellular Acidification

The stratum corneum (SC), the outermost epidermal layer, consists of nonviable anuclear keratinocytes, called corneocytes, which function as a protective barrier. The exact modes of cell death executed by keratinocytes of the upper stratum granulosum (SG1 cells) remain largely unknown. Here, using intravital imaging combined with intracellular Ca2+- and pH-responsive fluorescent probes, we aimed to dissect the SG1 death process in vivo. We found that SG1 cell death was preceded by prolonged (∼60 min) Ca2+ elevation and rapid induction of intracellular acidification. Once such intracellular ionic changes were initiated, they became sustained, irreversibly committing the SG1 cells to corneocyte conversion. Time-lapse imaging of isolated murine SG1 cells revealed that intracellular acidification was essential for the degradation of keratohyalin granules and nuclear DNA, phenomena specific to SC corneocyte formation. Furthermore, intravital imaging showed that the number of SG1 cells exhibiting Ca2+ elevation and the timing of intracellular acidification were both tightly regulated by the transient receptor potential cation channel V3. The functional activity of this protein was confirmed in isolated SG1 cells using whole-cell patch-clamp analysis. These findings provide a theoretical framework for improved understanding of the unique molecular mechanisms underlying keratinocyte-specific death mode, namely corneoptosis.

scholarly journals miR-182-5p and miR-378a-3p regulate ferroptosis in I/R-induced renal injury

2020 ◽  
Vol 11 (10) ◽  
Author(s):  
Chenguang Ding ◽  
Xiaoming Ding ◽  
Jin Zheng ◽  
Bo Wang ◽  
Yang Li ◽  
...  
Keyword(s):  
Cell Death ◽  
Renal Injury ◽  
Molecular Mechanisms ◽  
Ischemia Reperfusion ◽  
Kidney Injury ◽  
Luciferase Reporter ◽  
Renal Tubular Cell ◽  
In Cells

Abstract Renal tubular cell death is the key factor of the pathogenesis of ischemia/reperfusion (I/R) kidney injury. Ferroptosis is a type of regulated cell death (RCD) found in various diseases. However, the underlying molecular mechanisms related to ferroptosis in renal I/R injury remain unclear. In the present study, we investigated the regulatory role of microRNAs on ferroptosis in I/R-induced renal injury. We established the I/R-induced renal injury model in rats, and H/R induced HK-2 cells injury in vitro. CCK-8 was used to measure cell viability. Fe2+ and ROS levels were assayed to evaluate the activation of ferroptosis. We performed RNA sequencing to profile the miRNAs expression in H/R-induced injury and ferroptosis. Western blot analysis was used to detect the protein expression. qRT-PCR was used to detect the mRNA and miRNA levels in cells and tissues. We further used luciferase reporter assay to verify the direct targeting effect of miRNA. We found that ischemia/reperfusion-induced ferroptosis in rat’s kidney. We identified that miR-182-5p and miR-378a-3p were upregulated in the ferroptosis and H/R-induced injury, and correlates reversely with glutathione peroxidases 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) expression in renal I/R injury tissues, respectively. In vitro studies showed that miR-182-5p and miR-378a-3p induced ferroptosis in cells. We further found that miR-182-5p and miR-378a-3p regulated the expression of GPX4 and SLC7A11 negatively by directly binding to the 3′UTR of GPX4 and SLC7A11 mRNA. In vivo study showed that silencing miR-182-5p and miR-378a-3p alleviated the I/R-induced renal injury in rats. In conclusion, we demonstrated that I/R induced upregulation of miR-182-5p and miR-378a-3p, leading to activation of ferroptosis in renal injury through downregulation of GPX4 and SLC7A11.

scholarly journals Growth hormone protects against radiotherapy-induced cell death

2002 ◽  
pp. 535-541 ◽  
Author(s):  
O Madrid ◽  
S Varea ◽  
I Sanchez-Perez ◽  
L Gomez-Garcia ◽  
E De Miguel ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Cell Death ◽  
Hormone Receptor ◽  
Growth Hormone Receptor ◽  
Molecular Mechanisms ◽  
Dna Double Strand Breaks ◽  
Cell Viability Assay ◽  
Viability Assay ◽  
Rat Growth

BACKGROUND: In vivo treatment with growth hormone reduces radiation-associated mortality. The molecular mechanisms underlying this effect are unknown. It has been described that increased sensitivity to ionising radiation can be due to defects in machinery involved in detection and/or repair of DNA double-strand breaks. OBJECTIVE: To study the mechanisms involved in growth hormone action on the increased survival in irradiated cells. MATERIALS AND METHODS: CHO-4 cells stably expressing the growth hormone receptor were used. A cell viability assay was carried out to analyse the increase in survival induced by growth hormone in irradiated cells. To investigate whether the DNA repair mechanism could be implicated in this effect we performed DNA reactivation assays using pHIV-LUC and pCMV-betagal plasmids as control. Identical studies were also conducted using the radiomimetic drug, bleomycin. RESULTS: Growth hormone protects CHO-4 cells from bleomycin- and radiation-induced cell death. In pHIV-LUC transfected cells, a time-dependent decrease in luciferase activity was observed after irradiation in the absence of growth hormone. However, cells pretreated with this hormone maintained reporter activity. When cells were transfected with irradiated pHIV-LUC plasmid, only the hormone-treated cells recovered the transcriptional activity. CONCLUSIONS: Growth hormone exerts a radioprotective effect in CHO-4 cells stably transfected with the complementary DNA for the rat growth hormone receptor. The radioprotection is triggered directly by the hormone and it is also observed with bleomycin. The increased survival in response to radiation and bleomycin treatment induced by growth hormone correlates with an enhanced ability of the cells to repair damaged DNA.

scholarly journals Ivermectin induces autophagy-mediated cell death through the AKT/mTOR signaling pathway in glioma cells

2019 ◽  
Vol 39 (12) ◽  
Author(s):  
Jingjing Liu ◽  
Hongsheng Liang ◽  
Chen Chen ◽  
Xiaoxing Wang ◽  
Faling Qu ◽  
...  
Keyword(s):  
Cell Death ◽  
Molecular Mechanisms ◽  
Anticancer Agent ◽  
Glioma Cells ◽  
Mtor Signaling ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Transmission Electron ◽  
Diphenyltetrazolium Bromide

Abstract Glioma is one of the most common types of primary brain tumors. Ivermectin (IVM), a broad-spectrum antiparasitic drug, has been identified as a novel anticancer agent due to its inhibitory effects on the proliferation of glioma cells in vitro and in vivo. However, the ability of IVM to induce autophagy and its role in glioma cell death remains unclear. The main objective of the present study was to explore autophagy induced by IVM in glioma U251 and C6 cells, and the deep underlying molecular mechanisms. In addition, we examined the effects of autophagy on apoptosis in glioma cells. In the present study, transmission electron microscopy (TEM), immunofluorescence, Western blot and immunohistochemistry were used to evaluate autophagy activated by IVM. Cell viability was measured by 3-(4,5-dimethylthiazol2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and colony formation assay. The apoptosis rate was detected by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Meanwhile, autophagy inhibition was achieved by using chloroquine (CQ). U251-derived xenografts were established for examination of IVM-induced autophagy on glioma in vivo. Taken together, the results of the present study showed that autophagy induced by IVM has a protective effect on cell apoptosis in vitro and in vivo. Mechanistically, IVM induced autophagy through AKT/mTOR signaling and induced energy impairment. Our findings show that IVM is a promising anticancer agent and may be a potential effective treatment for glioma cancers.

scholarly journals Current translational potential and underlying molecular mechanisms of necroptosis

2019 ◽  
Vol 10 (11) ◽  
Author(s):  
Tamás Molnár ◽  
Anett Mázló ◽  
Vera Tslaf ◽  
Attila Gábor Szöllősi ◽  
Gabriella Emri ◽  
...  
Keyword(s):  
Cell Death ◽  
Protein Kinase ◽  
Posttranslational Modifications ◽  
Molecular Mechanisms ◽  
Inflammatory Diseases ◽  
Immune Surveillance ◽  
Kinase Domain ◽  
Tumor Formation

Abstract Cell death has a fundamental impact on the evolution of degenerative disorders, autoimmune processes, inflammatory diseases, tumor formation and immune surveillance. Over the past couple of decades extensive studies have uncovered novel cell death pathways, which are independent of apoptosis. Among these is necroptosis, a tightly regulated, inflammatory form of cell death. Necroptosis contribute to the pathogenesis of many diseases and in this review, we will focus exclusively on necroptosis in humans. Necroptosis is considered a backup mechanism of apoptosis, but the in vivo appearance of necroptosis indicates that both caspase-mediated and caspase-independent mechanisms control necroptosis. Necroptosis is regulated on multiple levels, from the transcription, to the stability and posttranslational modifications of the necrosome components, to the availability of molecular interaction partners and the localization of receptor-interacting serine/threonine-protein kinase 1 (RIPK1), receptor-interacting serine/threonine-protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). Accordingly, we classified the role of more than seventy molecules in necroptotic signaling based on consistent in vitro or in vivo evidence to understand the molecular background of necroptosis and to find opportunities where regulating the intensity and the modality of cell death could be exploited in clinical interventions. Necroptosis specific inhibitors are under development, but >20 drugs, already used in the treatment of various diseases, have the potential to regulate necroptosis. By listing necroptosis-modulated human diseases and cataloging the currently available drug-repertoire to modify necroptosis intensity, we hope to kick-start approaches with immediate translational potential. We also indicate where necroptosis regulating capacity should be considered in the current applications of these drugs.

scholarly journals Dominant-negative ATF5 rapidly depletes survivin in tumor cells

2019 ◽  
Vol 10 (10) ◽  
Author(s):  
Xiaotian Sun ◽  
James M. Angelastro ◽  
David Merino ◽  
Qing Zhou ◽  
Markus D. Siegelin ◽  
...  
Keyword(s):  
Cell Death ◽  
Tumor Cell ◽  
Cancer Cells ◽  
Dominant Negative ◽  
Tumor Cell Death ◽  
Cell Penetrating ◽  
Selective Death ◽  
Tumor Types

Abstract Survivin (BIRC5, product of the BIRC5 gene) is highly expressed in many tumor types and has been widely identified as a potential target for cancer therapy. However, effective anti-survivin drugs remain to be developed. Here we report that both vector-delivered and cell-penetrating dominant-negative (dn) forms of the transcription factor ATF5 that promote selective death of cancer cells in vitro and in vivo cause survivin depletion in tumor cell lines of varying origins. dn-ATF5 decreases levels of both survivin mRNA and protein. The depletion of survivin protein appears to be driven at least in part by enhanced proteasomal turnover and depletion of the deubiquitinase USP9X. Survivin loss is rapid and precedes the onset of cell death triggered by dn-ATF5. Although survivin downregulation is sufficient to drive tumor cell death, survivin over-expression does not rescue cancer cells from dn-ATF5-promoted apoptosis. This indicates that dn-ATF5 kills malignant cells by multiple mechanisms that include, but are not limited to, survivin depletion. Cell-penetrating forms of dn-ATF5 are currently being developed for potential therapeutic use and the present findings suggest that they may pose an advantage over treatments that target only survivin.

scholarly journals Iron toxicity, lipid peroxidation and ferroptosis after intracerebral haemorrhage

2019 ◽  
Vol 4 (2) ◽  
pp. 93-95 ◽  
Author(s):  
Jieru Wan ◽  
Honglei Ren ◽  
Jian Wang
Keyword(s):  
Lipid Peroxidation ◽  
Cell Death ◽  
Intracerebral Haemorrhage ◽  
Neuronal Death ◽  
Molecular Mechanisms ◽  
Apoptotic Cell ◽  
Iron Toxicity ◽  
Secondary Brain Injury

Intracerebral haemorrhage (ICH) is a devastating type of stroke with high mortality and morbidity. However, we have few options for ICH therapy and limited knowledge about post-ICH neuronal death and related mechanisms. In the aftermath of ICH, iron overload within the perihaematomal region can induce lethal reactive oxygen species (ROS) production and lipid peroxidation, which contribute to secondary brain injury. Indeed, iron chelation therapy has shown efficacy in preclinical ICH studies. Recently, an iron-dependent form of non-apoptotic cell death known as ferroptosis was identified. It is characterised by an accumulation of iron-induced lipid ROS, which leads to intracellular oxidative stress. The ROS cause damage to nucleic acids, proteins and lipid membranes, and eventually cell death. Recently, we and others discovered that ferroptosis does occur after haemorrhagic stroke in vitro and in vivo and contributes to neuronal death. Inhibition of ferroptosis is beneficial in several in vivo and in vitro ICH conditions. This minireview summarises current research on iron toxicity, lipid peroxidation and ferroptosis in the pathomechanisms of ICH, the underlying molecular mechanisms of ferroptosis and the potential for combined therapeutic strategies. Understanding the role of ferroptosis after ICH will provide a vital foundation for cell death-based ICH treatment and prevention.

Deletion of the Impg2 gene causes the degeneration of rod and cone cells in mice

2020 ◽  
Vol 29 (10) ◽  
pp. 1624-1634
Author(s):  
Huijuan Xu ◽  
Chao Qu ◽  
Li Gan ◽  
Kuanxiang Sun ◽  
Junkai Tan ◽  
...  
Keyword(s):  
Cell Death ◽  
Er Stress ◽  
Molecular Mechanisms ◽  
Histopathological Examination ◽  
Apoptotic Cell ◽  
Macular Dystrophy ◽  
Cone Cell ◽  
Rod Cells ◽  
Cone Cells

Abstract Variants in interphotoreceptor matrix proteoglycans (IMPG2) have been reported in retinitis pigmentosa (RP) and vitelliform macular dystrophy (VMD) patients. However, the underlying molecular mechanisms remain elusive due to a lack of suitable disease models. We developed two independent Impg2 knockout (KO) mouse models using the CRISPR/Cas9 technique to assess the in vivo functions of Impg2 in the retina. Impg2 ablation in mice recapitulated the RP phenotypes of patients, including an attenuated electroretinogram (ERG) response and the progressive degeneration of photoreceptors. The histopathological examination of Impg2-KO mice revealed irregularly arranged rod cells and mislocalized rhodopsin protein in the inner segment at 6 months of age. In addition to the pathological changes in rod cells, cone cells were also affected in KO retinas. KO retinas exhibited progressive cone cell death and impaired cone cell elongation. Further immunoblotting analysis revealed increased levels of endoplasmic reticulum (ER) stress-related proteins, including C/EBP homologous protein (CHOP), immunoglobulin heavy-chain-binding protein (BIP) and protein disulfide isomerase (PDI), in Impg2-KO mouse retinas. Increased gliosis and apoptotic cell death were also observed in the KO retinas. As autophagy is closely associated with ER stress, we then checked whether autophagy was disturbed in Impg2-KO mouse retinas. The results showed that autophagy was impaired in KO retinas, as revealed by the increased accumulation of SQSTM1 and other proteins involved in autophagy. Our results demonstrate the essential roles of Impg2 in the retina, and this study provides novel models for mechanistic investigations and development of therapies for RP caused by IMPG2 mutations.

Molecular mechanisms of activated T cell death in vivo

2002 ◽  
Vol 14 (3) ◽  
pp. 354-359 ◽  
Author(s):  
David A Hildeman ◽  
Yanan Zhu ◽  
Thomas C Mitchell ◽  
John Kappler ◽  
Philippa Marrack
Keyword(s):  
Cell Death ◽  
T Cell ◽  
Molecular Mechanisms ◽  
Activated T Cell

scholarly journals A model of muscle atrophy based on live microscopy of muscle remodelling in Drosophila metamorphosis

2016 ◽  
Vol 3 (2) ◽  
pp. 150517 ◽  
Author(s):  
Yadav Kuleesha ◽  
Wee Choo Puah ◽  
Martin Wasser
Keyword(s):  
Muscle Atrophy ◽  
Cell Biology ◽  
Muscle Wasting ◽  
Cathepsin L ◽  
Muscle Growth ◽  
Muscle Size ◽  
Abdominal Muscles ◽  
Quantitative Image ◽  
Muscle Remodelling

Genes controlling muscle size and survival play important roles in muscle wasting diseases. In Drosophila melanogaster metamorphosis, larval abdominal muscles undergo two developmental fates. While a doomed population is eliminated by cell death, another persistent group is remodelled and survives into adulthood. To identify and characterize genes involved in the development of remodelled muscles, we devised a workflow consisting of in vivo imaging, targeted gene perturbation and quantitative image analysis. We show that inhibition of TOR signalling and activation of autophagy promote developmental muscle atrophy in early, while TOR and yorkie activation are required for muscle growth in late pupation. We discovered changes in the localization of myonuclei during remodelling that involve anti-polar migration leading to central clustering followed by polar migration resulting in localization along the midline. We demonstrate that the Cathepsin L orthologue Cp1 is required for myonuclear clustering in mid, while autophagy contributes to central positioning of nuclei in late metamorphosis. In conclusion, studying muscle remodelling in metamorphosis can provide new insights into the cell biology of muscle wasting.

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