scholarly journals Robustness of epithelial sealing is an emerging property of local ERK feedbacks driven by cell elimination

2020 ◽  
Author(s):  
Léo Valon ◽  
Anđela Davidović ◽  
Florence Levillayer ◽  
Mathilde Chouly ◽  
Fabiana Cerqueira-Campos ◽  
...  
Keyword(s):  
Cell Death ◽  
Single Layer ◽  
Temporal Distribution ◽  
High Rate ◽  
Caspase Activation ◽  
Epithelial Homeostasis ◽  
Transient Activation ◽  
Spatio Temporal ◽  
Cell Extrusion

AbstractWhile the pathways regulating apoptosis and cell extrusion are rather well described1,2, what regulates the precise spatio-temporal distribution of cell elimination in tissues remains largely unknown. This is particularly relevant for epithelia with high rates of cell elimination, a widespread situation during embryogenesis3–6 and epithelial homeostasis7, where concomitant death of neighbours could impair the maintenance of epithelial sealing. However, the extent to which epithelial tissues can cope with concomitant cell death, and whether any mechanism regulates such occurrence have never been explored so far. Here, using the Drosophila pupal notum (a single layer epithelium) and a new optogenetic tool to trigger caspase activation and cell extrusion, we first show that concomitant death of clusters of at least three cells is sufficient to transiently impair epithelial sealing. Such clustered extrusion was almost never observed in vivo, suggesting the existence of a mechanism preventing concomitant elimination of neighbours. Statistical analysis and simulations of cell death distribution in the notum highlighted a transient and local protective phase occurring near every dying cell. This protection is driven by a transient activation of ERK in the direct neighbours of extruding cells which reverts caspase activation and prevents elimination of cells in clusters. Altogether, this study demonstrates that the distribution of cell elimination in epithelia is an emerging property of transient and local feedbacks through ERK activation which is required to maintain epithelial sealing in conditions of high rate of cell elimination.

scholarly journals Dynamics of in vivo ASC speck formation

2017 ◽  
Vol 216 (9) ◽  
pp. 2891-2909 ◽  
Author(s):  
Paola Kuri ◽  
Nicole L. Schieber ◽  
Thomas Thumberger ◽  
Joachim Wittbrodt ◽  
Yannick Schwab ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Death ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Inflammasome Activation ◽  
Caspase Activation ◽  
Endogenous Gene ◽  
Pyrin Domain ◽  
Effector Caspase

Activated danger or pathogen sensors trigger assembly of the inflammasome adaptor ASC into specks, large signaling platforms considered hallmarks of inflammasome activation. Because a lack of in vivo tools has prevented the study of endogenous ASC dynamics, we generated a live ASC reporter through CRISPR/Cas9 tagging of the endogenous gene in zebrafish. We see strong ASC expression in the skin and other epithelia that act as barriers to insult. A toxic stimulus triggered speck formation and rapid pyroptosis in keratinocytes in vivo. Macrophages engulfed and digested that speck-containing, pyroptotic debris. A three-dimensional, ultrastructural reconstruction, based on correlative light and electron microscopy of the in vivo assembled specks revealed a compact network of highly intercrossed filaments, whereas pyrin domain (PYD) or caspase activation and recruitment domain alone formed filamentous aggregates. The effector caspase is recruited through PYD, whose overexpression induced pyroptosis but only after substantial delay. Therefore, formation of a single, compact speck and rapid cell-death induction in vivo requires a full-length ASC.

scholarly journals Microtubule disassembly by caspases is the rate-limiting step of cell extrusion

2021 ◽  
Author(s):  
Alexis Villars ◽  
Alexis Matamoro-Vidal ◽  
Florence Levillayer ◽  
Romain Levayer
Keyword(s):  
Epithelial Cell ◽  
Single Layer ◽  
General Function ◽  
Caspase Activation ◽  
Apical Constriction ◽  
Rate Limiting Step ◽  
Cellular Factors ◽  
Effector Caspases ◽  
Rate Limiting ◽  
Cell Extrusion

Epithelial cell death is essential for tissue homeostasis, robustness and morphogenesis. The expulsion of epithelial cells following caspase activation requires well-orchestrated remodeling steps leading to cell elimination without impairing tissue sealing. While numerous studies have provided insight about the process of cell extrusion, we still know very little about the relationship between caspase activation and the remodeling steps of cell extrusion. Moreover, most studies of cell extrusion focused on the regulation of actomyosin and steps leading to the formation of a supracellular contractile ring. However, the contribution of other cellular factors to cell extrusion has been poorly explored. Using the Drosophila pupal notum, a single layer epithelium where most extrusion events are caspase-dependent, we first showed that the initiation of cell extrusion and apical constriction are surprisingly not associated with the modulation of actomyosin concentration/dynamics. Instead, cell apical constriction is initiated by the disassembly of a medio-apical mesh of microtubules which is driven by effector caspases. We confirmed that local and rapid increase/decrease of microtubules is sufficient to respectively expand/constrict cell apical area. Importantly, the depletion of microtubules is sufficient to bypass the requirement of caspases for cell extrusion. This study shows that microtubules disassembly by caspases is a key rate-limiting steps of extrusion, and outlines a more general function of microtubules in epithelial cell shape stabilisation.

scholarly journals Inhibiting Myosin Light Chain Kinase Induces Apoptosis In Vitro and In Vivo

2005 ◽  
Vol 25 (14) ◽  
pp. 6259-6266 ◽  
Author(s):  
Fabeha Fazal ◽  
Lianzhi Gu ◽  
Ivanna Ihnatovych ◽  
YooJeong Han ◽  
WenYang Hu ◽  
...  
Keyword(s):  
Cell Death ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Apoptotic Cells ◽  
Caspase Activation ◽  
Inhibitory Antibody ◽  
Necrosis Factor Alpha

ABSTRACT Previous short-term studies have correlated an increase in the phosphorylation of the 20-kDa light chain of myosin II (MLC20) with blebbing in apoptotic cells. We have found that this increase in MLC20 phosphorylation is rapidly followed by MLC20 dephosphorylation when cells are stimulated with various apoptotic agents. MLC20 dephosphorylation is not a consequence of apoptosis because MLC20 dephosphorylation precedes caspase activation when cells are stimulated with a proapoptotic agent or when myosin light chain kinase (MLCK) is inhibited pharmacologically or by microinjecting an inhibitory antibody to MLCK. Moreover, blocking caspase activation increased cell survival when MLCK is inhibited or when cells are treated with tumor necrosis factor alpha. Depolymerizing actin filaments or detaching cells, processes that destabilize the cytoskeleton, or inhibiting myosin ATPase activity also resulted in MLC20 dephosphorylation and cell death. In vivo experiments showed that inhibiting MLCK increased the number of apoptotic cells and retarded the growth of mammary cancer cells in mice. Thus, MLC20 dephosphorylation occurs during physiological cell death and prolonged MLC20 dephosphorylation can trigger apoptosis.

scholarly journals Proapoptotic RHG genes and mitochondria play a key non-apoptotic role in remodelling the Drosophila sensory system

2021 ◽  
Author(s):  
Amrita Mukherjee ◽  
Sinziana Pop ◽  
Shu Kondo ◽  
Darren W Williams
Keyword(s):  
Cell Death ◽  
Cell Fate ◽  
Sensory Neuron ◽  
Sensory Neurons ◽  
Molecular Mechanisms ◽  
Sensory System ◽  
Caspase Activation ◽  
Effector Caspase ◽  
Effector Caspases

AbstractCaspases are best known for their role in programmed cell death but have also been found to be important in several non-apoptotic phenomena such as cell fate specification, cell migration and terminal differentiation. The dynamics of such sub-lethal caspase events and the molecular mechanisms regulating them are still largely unknown. As more tools for visualizing and manipulating caspase activation in vivo become available, greater insights into this biology are being made. Using a new and sensitive in vivo effector caspase probe, called SR4VH, we demonstrate that effector caspases are activated in pruning sensory neurons earlier than previously thought and that the level of caspase activation in these neurons is consistently lower than in neurons undergoing cell death. We reveal that Grim and Reaper, two of the four pro-apoptotic RHG proteins, are required for sensory neuron pruning and that disrupting the dynamics of the mitochondrial network prevents effector caspase activation in both pruning and dying sensory neurons. Overall, our findings demonstrate that a sublethal deployment of the ‘apoptotic machinery’ is critical for remodelling dendrites and also reveal a direct link between mitochondria and sensory neuron cell death in vivo.

scholarly journals Spatio-temporal activation of caspase revealed by indicator that is insensitive to environmental effects

2003 ◽  
Vol 160 (2) ◽  
pp. 235-243 ◽  
Author(s):  
Kiwamu Takemoto ◽  
Takeharu Nagai ◽  
Atsushi Miyawaki ◽  
Masayuki Miura
Keyword(s):  
Caspase 3 ◽  
Fluorescent Protein ◽  
Morphological Changes ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy ◽  
Caspase Activation ◽  
Caspase 9 ◽  
Enhanced Yellow Fluorescent Protein ◽  
Spatio Temporal

Indicator molecules for caspase-3 activation have been reported that use fluorescence resonance energy transfer (FRET) between an enhanced cyan fluorescent protein (the donor) and enhanced yellow fluorescent protein (EYFP; the acceptor). Because EYFP is highly sensitive to proton (H+) and chloride ion (Cl−) levels, which can change during apoptosis, this indicator's ability to trace the precise dynamics of caspase activation is limited, especially in vivo. Here, we generated an H+- and Cl−-insensitive indicator for caspase activation, SCAT, in which EYFP was replaced with Venus, and monitored the spatio-temporal activation of caspases in living cells. Caspase-3 activation was initiated first in the cytosol and then in the nucleus, and rapidly reached maximum activation in 10 min or less. Furthermore, the nuclear activation of caspase-3 preceded the nuclear apoptotic morphological changes. In contrast, the completion of caspase-9 activation took much longer and its activation was attenuated in the nucleus. However, the time between the initiation of caspase-9 activation and the morphological changes was quite similar to that seen for caspase-3, indicating the activation of both caspases occurred essentially simultaneously during the initiation of apoptosis.

scholarly journals Altered Cytochrome c Display Precedes Apoptotic Cell Death in Drosophila

1999 ◽  
Vol 144 (4) ◽  
pp. 701-710 ◽  
Author(s):  
Johnson Varkey ◽  
Po Chen ◽  
Ronald Jemmerson ◽  
John M. Abrams
Keyword(s):  
Cell Death ◽  
Cytochrome C ◽  
Apoptotic Cell ◽  
In Vitro Models ◽  
Caspase Activity ◽  
Caspase Activation ◽  
Conditional Expression ◽  
Necessary And Sufficient

Drosophila affords a genetically well-defined system to study apoptosis in vivo. It offers a powerful extension to in vitro models that have implicated a requirement for cytochrome c in caspase activation and apoptosis. We found that an overt alteration in cytochrome c anticipates programmed cell death (PCD) in Drosophila tissues, occurring at a time that considerably precedes other known indicators of apoptosis. The altered configuration is manifested by display of an otherwise hidden epitope and occurs without release of the protein into the cytosol. Conditional expression of the Drosophila death activators, reaper or grim, provoked apoptogenic cytochrome c display and, surprisingly, caspase activity was necessary and sufficient to induce this alteration. In cell-free studies, cytosolic caspase activation was triggered by mitochondria from apoptotic cells but identical preparations from healthy cells were inactive. Our observations provide compelling validation of an early role for altered cytochrome c in PCD and suggest propagation of apoptotic physiology through reciprocal, feed-forward amplification involving cytochrome c and caspases.

scholarly journals Local initiation of caspase activation in Drosophila salivary gland programmed cell death in vivo

2007 ◽  
Vol 104 (33) ◽  
pp. 13367-13372 ◽  
Author(s):  
K. Takemoto ◽  
E. Kuranaga ◽  
A. Tonoki ◽  
T. Nagai ◽  
A. Miyawaki ◽  
...  
Keyword(s):  
Cell Death ◽  
Salivary Gland ◽  
Programmed Cell Death ◽  
Caspase Activation

scholarly journals Hypoxia Enhanced Glioblastoma Resistance to Erastin-Induced Ferroptosis by Up-Regulating GPX4 via PI3K/AKT/HIF-1α Axis

2021 ◽  
Author(s):  
Shicheng Sun ◽  
Changfa Guo ◽  
Bo Pang ◽  
Taihong Gao ◽  
Xiangsheng Su ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
High Rate ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Akt Inhibitor ◽  
Orthotopic Xenograft ◽  
Subcutaneous Xenograft ◽  
U251 Cells

Abstract Background Glioblastoma is the deadliest type of primary brain tumor with a high rate of recurrence and treatment resistance. Hypoxia contributed much to radiotherapy resistance and chemoresistance of cancer. Ferroptosis is a nonapoptotic, oxidative cell death and identified as a potential anticancer mechanism in recent years. Erastin acts as a ferroptosis activator and shows a potential role in tumor treatment but the relationship between hypoxia and erastin resistance in glioblastoma has not been explained. This study aimed to investigate the role and underlying mechanism of hypoxia in erastin-induced ferroptosis in glioblastoma. Methods Cell proliferation and viability were determined by Cell Counting Kit-8 (CCK-8) assay, flow cytometry, TUNEL assays, and clone formation assay. Lipid peroxides level was analyzed by Malondialdehyde (MDA) assay and flow cytometry using C11-BODIPY dye. The correlation between HIF-1α and GPX4 expression was detected in data from the TCGA database and was determined by ChIP-qPCR assay and luciferase reporter assay. Subcutaneous xenograft and orthotopic xenograft models were established to test our findings in vivo. Results Hypoxia for at least 16 hours significantly suppressed erastin-induced ferroptosis by up-regulating glutathione peroxidase 4 (GPX4) expression in U87 and U251 cells. Hypoxia promotes GPX4 expression via enhancing the PI3K/AKT/HIF-1α pathway. Mechanistically, HIF-1α directly bound to the GPX4 gene promoter region and promoted GPX4 transcription. AKT inhibitor MK-2206 and HIF-1α inhibitor PX-478 could significantly reverse the effect. Besides, under normoxia, PX-478 could induce a higher lipid peroxidation level by decreasing GPX4 expression in U87 and U251 cells but cannot induced cell death directly, and it could significantly enhance the tumor cell killing effect of erastin. In vivo, combination of PX-478 and erastin had a coordinated intensification effect on anticancer activity uncovered by subcutaneous xenograft and orthotopic xenograft mouse models. Conclusions Hypoxia enhanced glioblastoma resistance to erastin-induced ferroptosis by activating PI3K/AKT/HIF-1α pathway and promoting GPX4 expression in a transcriptional regulation way. Combination therapy of PX-478 and erastin may be a potential strategy against glioblastoma.

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