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 A Role for H2O2 and TRPM2 in the Induction of Cell Death: Studies in KGN Cells

Antioxidants ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 518 ◽  
Author(s):  
Carsten Theo Hack ◽  
Theresa Buck ◽  
Konstantin Bagnjuk ◽  
Katja Eubler ◽  
Lars Kunz ◽  
...  
Keyword(s):  
Cell Death ◽  
Transient Receptor Potential ◽  
Therapeutic Potential ◽  
Apoptotic Cell ◽  
Receptor Potential ◽  
Granulosa Cell Tumor ◽  
Nadph Oxidase 4 ◽  
Transient Receptor Potential Melastatin ◽  
Trpm2 Channel

Recent studies showed that KGN cells, derived from a human granulosa cell tumor (GCT), express NADPH oxidase 4 (NOX4), an important source of H2O2. Transient receptor potential melastatin 2 (TRPM2) channel is a Ca2+ permeable cation channel that can be activated by H2O2 and plays an important role in cellular functions. It is also able to promote susceptibility to cell death. We studied expression and functionality of TRPM2 in KGN cells and examined GCT tissue microarrays (TMAs) to explore in vivo relevance. We employed live cell, calcium and mitochondrial imaging, viability assays, fluorescence activated cell sorting (FACS) analysis, Western blotting and immunohistochemistry. We confirmed that KGN cells produce H2O2 and found that they express functional TRPM2. H2O2 increased intracellular Ca2+ levels and N-(p-Amylcinnamoyl)anthranilic acid (ACA), a TRPM2 inhibitor, blocked this action. H2O2 caused mitochondrial fragmentation and apoptotic cell death, which could be attenuated by a scavenger (Trolox). Immunohistochemistry showed parallel expression of NOX4 and TRPM2 in all 73 tumor samples examined. The results suggest that GCTs can be endowed with a system that may convey susceptibility to cell death. If so, induction of oxidative stress may be beneficial in GCT therapy. Our results also imply a therapeutic potential for TRPM2 as a drug target in GCTs.

Cell death modulation by transient receptor potential melastatin channels TRPM2 and TRPM7 and their underlying molecular mechanisms

2021 ◽  
pp. 114664
Author(s):  
Ruixue Shi ◽  
Yu Fu ◽  
Dongyi Zhao ◽  
Tomasz Boczek ◽  
Wuyang Wang ◽  
...  
Keyword(s):  
Cell Death ◽  
Molecular Mechanisms ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Transient Receptor Potential Melastatin ◽  
Transient Receptor

scholarly journals Quantitative intravital imaging reveals in vivo dynamics of physiological-stress induced mitophagy

2020 ◽  
Author(s):  
Paul J. Wrighton ◽  
Arkadi Shwartz ◽  
Jin-Mi Heo ◽  
Eleanor D. Quenzer ◽  
Kyle A. LaBella ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Cultured Cells ◽  
Physiological Stress ◽  
Hypoxia Inducible Factor ◽  
Imaging Study ◽  
Time Lapse ◽  
Intravital Imaging ◽  
Response Dynamics ◽  
Pathway Activation

ABSTRACTMitophagy, the selective recycling of mitochondria through autophagy, is a crucial metabolic process induced by cellular stress, and defects are linked to aging, sarcopenia, and neurodegenerative diseases. To therapeutically target mitophagy, the fundamental in vivo dynamics and molecular mechanisms must be fully understood. Here, we generated mitophagy biosensor zebrafish lines expressing mitochondrially targeted, pH-sensitive, fluorescent probes mito-Keima and mito-EGFP-mCherry and used quantitative intravital imaging to illuminate mitophagy during physiological stresses—embryonic development, fasting and hypoxia. In fasted muscle, volumetric mitolysosome size analyses documented organelle stress-response dynamics, and time-lapse imaging revealed mitochondrial filaments undergo piecemeal fragmentation and recycling rather than the wholesale turnover observed in cultured cells. Hypoxia-inducible factor (Hif) pathway activation through physiological hypoxia or chemical or genetic modulation also provoked mitophagy. Intriguingly, mutation of a single mitophagy receptor bnip3 prevented this effect, whereas disruption of other putative hypoxia-associated mitophagy genes bnip3la (nix), fundc1, pink1 or prkn (Parkin) had no effect. This in vivo imaging study establishes fundamental dynamics of fasting-induced mitophagy and identifies bnip3 as the master regulator of Hif-induced mitophagy in vertebrate muscle.

scholarly journals Genetically encoded cell-death indicators (GEDI) to detect an early irreversible commitment to neurodegeneration

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jeremy W. Linsley ◽  
Kevan Shah ◽  
Nicholas Castello ◽  
Michelle Chan ◽  
Dominik Haddad ◽  
...  
Keyword(s):  
Cell Death ◽  
Time Lapse ◽  
Death Process ◽  
Cell Detection ◽  
High Throughput Analysis ◽  
Time Resolved ◽  
Single Cell Detection ◽  
Human Neurons

AbstractCell death is a critical process that occurs normally in health and disease. However, its study is limited due to available technologies that only detect very late stages in the process or specific death mechanisms. Here, we report the development of a family of fluorescent biosensors called genetically encoded death indicators (GEDIs). GEDIs specifically detect an intracellular Ca2+ level that cells achieve early in the cell death process and that marks a stage at which cells are irreversibly committed to die. The time-resolved nature of a GEDI delineates a binary demarcation of cell life and death in real time, reformulating the definition of cell death. We demonstrate that GEDIs acutely and accurately report death of rodent and human neurons in vitro, and show that GEDIs enable an automated imaging platform for single cell detection of neuronal death in vivo in zebrafish larvae. With a quantitative pseudo-ratiometric signal, GEDIs facilitate high-throughput analysis of cell death in time-lapse imaging analysis, providing the necessary resolution and scale to identify early factors leading to cell death in studies of neurodegeneration.

scholarly journals Genetically encoded cell-death indicators (GEDI) to detect an early irreversible commitment to neurodegeneration

2019 ◽  
Author(s):  
Jeremy W. Linsley ◽  
Kevan Shah ◽  
Nicholas Castello ◽  
Michelle Chan ◽  
Dominic Haddad ◽  
...  
Keyword(s):  
Cell Death ◽  
Time Lapse ◽  
Death Process ◽  
Cell Detection ◽  
High Throughput Analysis ◽  
Time Resolved ◽  
Single Cell Detection ◽  
Human Neurons

AbstractCell death is a critical process that occurs normally in health and disease. However, its study is limited due to available technologies that only detect very late stages in the process or specific death mechanisms. Here, we report the development of a new fluorescent biosensor called genetically encoded death indicator (GEDI). GEDI specifically detects an intracellular Ca2+ level that cells achieve early in the cell death process and marks a stage at which cells are irreversibly committed to die. The time-resolved nature of GEDI delineates a binary demarcation of cell life and death in real time, reformulating the definition of cell death. We demonstrate that GEDI acutely and accurately reports death of rodent and human neurons in vitro, and show GEDI enables a novel automated imaging platform for single cell detection of neuronal death in vivo in zebrafish larvae. With a quantitative pseudo-ratiometric signal, GEDI facilitates high-throughput analysis of cell death in time lapse imaging analysis, providing the necessary resolution and scale to identify early factors leading to cell death in studies of neurodegeneration.

scholarly journals Upregulation of Na+ and Ca2+ transporters in arterial smooth muscle from ouabain-induced hypertensive rats

2010 ◽  
Vol 298 (1) ◽  
pp. H263-H274 ◽  
Author(s):  
Maria V. Pulina ◽  
Alessandra Zulian ◽  
Roberto Berra-Romani ◽  
Olga Beskina ◽  
Amparo Mazzocco-Spezzia ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Smooth Muscle ◽  
Molecular Mechanisms ◽  
Transient Receptor Potential ◽  
Protein A ◽  
Receptor Potential ◽  
Atpase Inhibitor ◽  
Rna Knockdown

Prolonged ouabain administration (25 μg·kg−1·day−1 for 5 wk) induces “ouabain hypertension” (OH) in rats, but the molecular mechanisms by which ouabain elevates blood pressure are unknown. Here, we compared Ca2+ signaling in mesenteric artery smooth muscle cells (ASMCs) from normotensive (NT) and OH rats. Resting cytosolic free Ca2+ concentration ([Ca2+]cyt; measured with fura-2) and phenylephrine-induced Ca2+ transients were augmented in freshly dissociated OH ASMCs. Immunoblots revealed that the expression of the ouabain-sensitive α2-subunit of Na+ pumps, but not the predominant, ouabain-resistant α1-subunit, was increased (2.5-fold vs. NT ASMCs) as was Na+/Ca2+ exchanger-1 (NCX1; 6-fold vs. NT) in OH arteries. Ca2+ entry, activated by sarcoplasmic reticulum (SR) Ca2+ store depletion with cyclopiazonic acid (SR Ca2+-ATPase inhibitor) or caffeine, was augmented in OH ASMCs. This reflected an augmented expression of 2.5-fold in OH ASMCs of C-type transient receptor potential TRPC1, an essential component of store-operated channels (SOCs); two other components of some SOCs were not expressed (TRPC4) or were not upregulated (TRPC5). Ba2+ entry activated by the diacylglycerol analog 1-oleoyl-2-acetyl- sn-glycerol [a measure of receptor-operated channel (ROC) activity] was much greater in OH than NT ASMCs. This correlated with a sixfold upregulation of TRPC6 protein, a ROC family member. Importantly, in primary cultured mesenteric ASMCs from normal rats, 72-h treatment with 100 nM ouabain significantly augmented NCX1 and TRPC6 protein expression and increased resting [Ca2+]cyt and ROC activity. SOC activity was also increased. Silencer RNA knockdown of NCX1 markedly downregulated TRPC6 and eliminated the ouabain-induced augmentation; silencer RNA knockdown of TRPC6 did not affect NCX1 expression but greatly attenuated its upregulation by ouabain. Clearly, NCX1 and TRPC6 expression are interrelated. Thus, prolonged ouabain treatment upregulates the Na+ pump α2-subunit-NCX1-TRPC6 (ROC) Ca2+ signaling pathway in arterial myocytes in vitro as well as in vivo. This may explain the augmented myogenic responses and enhanced phenylephrine-induced vasoconstriction in OH arteries ( 83 ) as well as the high blood pressure in OH rats.

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 Quantitative intravital imaging in zebrafish reveals in vivo dynamics of physiological-stress-induced mitophagy

2021 ◽  
Vol 134 (4) ◽  
pp. jcs256255
Author(s):  
Paul J. Wrighton ◽  
Arkadi Shwartz ◽  
Jin-Mi Heo ◽  
Eleanor D. Quenzer ◽  
Kyle A. LaBella ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Cultured Cells ◽  
Physiological Stress ◽  
Hypoxia Inducible Factor ◽  
Imaging Study ◽  
Time Lapse ◽  
Intravital Imaging ◽  
Response Dynamics ◽  
Pathway Activation

ABSTRACTMitophagy, the selective recycling of mitochondria through autophagy, is a crucial metabolic process induced by cellular stress, and defects are linked to aging, sarcopenia and neurodegenerative diseases. To therapeutically target mitophagy, the fundamental in vivo dynamics and molecular mechanisms must be fully understood. Here, we generated mitophagy biosensor zebrafish lines expressing mitochondrially targeted, pH-sensitive fluorescent probes, mito-Keima and mito-EGFP–mCherry, and used quantitative intravital imaging to illuminate mitophagy during physiological stresses, namely, embryonic development, fasting and hypoxia. In fasted muscle, volumetric mitolysosome size analyses documented organelle stress response dynamics, and time-lapse imaging revealed that mitochondrial filaments undergo piecemeal fragmentation and recycling rather than the wholesale turnover observed in cultured cells. Hypoxia-inducible factor (Hif) pathway activation through physiological hypoxia or chemical or genetic modulation also provoked mitophagy. Intriguingly, mutation of a single mitophagy receptor (bnip3) prevented this effect, whereas disruption of other putative hypoxia-associated mitophagy genes [bnip3la (nix), fundc1, pink1 or prkn (Parkin)] had no effect. This in vivo imaging study establishes fundamental dynamics of fasting-induced mitophagy and identifies bnip3 as the master regulator of Hif-induced mitophagy in vertebrate muscle.

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