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 Dynamics of ASC speck formation during skin inflammatory responses in vivo

2017 ◽  
Author(s):  
Paola Kuri ◽  
Nicole L. Schieber ◽  
Thomas Thumberger ◽  
Joachim Wittbrodt ◽  
Yannick Schwab ◽  
...  
Keyword(s):  
Cell Death ◽  
Real Time ◽  
Inflammatory Responses ◽  
Full Length ◽  
Inflammasome Activation ◽  
Endogenous Gene ◽  
Effector Caspase

AbstractActivated danger or pathogen sensors trigger assembly of the inflammasome adaptor ASC into specks, large signalling 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 this speck-containing pyroptotic debris. A 3D ultrastructural reconstruction based on CLEM of in vivo assembled specks revealed a compact network of highly intercrossed filaments, whereas PYD or CARD alone formed filamentous aggregates. The effector caspase is recruited through PYD, whose overexpression induced pyroptosis, but after substantial delay. Therefore, formation of a single compact speck and rapid cell death induction in vivo requires full-length ASC.One Sentence SummaryWith a new endogenous ASC real-time reporter we characterize speck dynamics in vivo as well as the concomitant pyroptosis speck formation causes in keratinocytes.

scholarly journals The Roles of Inflammasomes in Host Defense against Mycobacterium tuberculosis

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 120
Author(s):  
Jialu Ma ◽  
Shasha Zhao ◽  
Xiao Gao ◽  
Rui Wang ◽  
Juan Liu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Host Defense ◽  
Inflammatory Responses ◽  
Inflammasome Activation ◽  
Caspase Activation ◽  
Nucleotide Binding Domain ◽  
Caspase 1 ◽  
Pyrin Domain

Mycobacterium tuberculosis (MTB) infection is characterized by granulomatous lung lesions and systemic inflammatory responses during active disease. Inflammasome activation is involved in regulation of inflammation. Inflammasomes are multiprotein complexes serving a platform for activation of caspase-1, which cleaves the proinflammatory cytokines such as interleukin-1β (IL-1β) and IL-18 into their active forms. These cytokines play an essential role in MTB control. MTB infection triggers activation of the nucleotide-binding domain, leucine-rich-repeat containing family, pyrin domain-containing 3 (NLRP3) and absent in melanoma 2 (AIM2) inflammasomes in vitro, but only AIM2 and apoptosis-associated speck-like protein containing a caspase-activation recruitment domain (ASC), rather than NLRP3 or caspase-1, favor host survival and restriction of mycobacterial replication in vivo. Interferons (IFNs) inhibits MTB-induced inflammasome activation and IL-1 signaling. In this review, we focus on activation and regulation of the NLRP3 and AIM2 inflammasomes after exposure to MTB, as well as the effect of inflammasome activation on host defense against the infection.

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.

Solving the mechanisms responsible for organelle behavior in vivo by same-cell correlative light and electron microscopy

1992 ◽  
Vol 50 (1) ◽  
pp. 14-15
Author(s):  
Conly L. Rieder
Keyword(s):  
Electron Microscopy ◽  
Quantitative Analysis ◽  
Light Microscopy ◽  
Living Cell ◽  
Light And Electron Microscopy ◽  
Time Behavior ◽  
Dynamic Interactions ◽  
Positive Identification ◽  
Cellular Components

The behavior of many cellular components, and their dynamic interactions, can be characterized in the living cell with considerable spatial and temporal resolution by video-enhanced light microscopy (video-LM). Indeed, under the appropriate conditions video-LM can be used to determine the real-time behavior of organelles ≤ 25-nm in diameter (e.g., individual microtubules—see). However, when pushed to its limit the structures and components observed within the cell by video-LM cannot be resolved nor necessarily even identified, only detected. Positive identification and a quantitative analysis often requires the corresponding electron microcopy (EM).

scholarly journals Ultrastructural Characterization of Flashing Mitochondria

Contact ◽  
2018 ◽  
Vol 1 ◽  
pp. 251525641880142
Author(s):  
Manon Rosselin ◽  
Paula Nunes-Hasler ◽  
Nicolas Demaurex
Keyword(s):  
Electron Microscopy ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Volume Ratio ◽  
Tubular Structures ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact

Mitochondria undergo spontaneous transient elevations in matrix pH associated with drops in mitochondrial membrane potential. These mitopHlashes require a functional respiratory chain and the profusion protein optic atrophy 1, but their mechanistic basis is unclear. To gain insight on the origin of these dynamic events, we resolved the ultrastructure of flashing mitochondria by correlative light and electron microscopy. HeLa cells expressing the matrix-targeted pH probe mitoSypHer were screened for mitopHlashes and fixed immediately after the occurrence of a flashing event. The cells were then processed for imaging by serial block face scanning electron microscopy using a focused ion beam to generate ∼1,200 slices of 10 nm thickness from a 28 µm × 15 µm cellular volume. Correlation of live/fixed fluorescence and electron microscopy images allowed the unambiguous identification of flashing and nonflashing mitochondria. Three-dimensional reconstruction and surface mapping revealed that each tomogram contained two flashing mitochondria of unequal sizes, one being much larger than the average mitochondrial volume. Flashing mitochondria were 10-fold larger than silent mitochondria but with a surface to volume ratio and a cristae volume similar to nonflashing mitochondria. Flashing mitochondria were connected by tubular structures, formed more membrane contact sites, and a constriction was observed at a junction between a flashing mitochondrion and a nonflashing mitochondrion. These data indicate that flashing mitochondria are structurally preserved and bioenergetically competent but form numerous membrane contact sites and are connected by tubular structures, consistent with our earlier suggestion that mitopHlashes might be triggered by the opening of fusion pores between contiguous mitochondria.

Abstract 530: Hemostasis Revisited: a Combined Light and Electron Microscopy Analysis of Hemostatic Plug Formation in vivo

2018 ◽  
Vol 38 (Suppl_1) ◽  
Author(s):  
Maurizio Tomaiuolo ◽  
Chelsea N Matzko ◽  
Izmarie Poventud-Fuentes ◽  
John W Weisel ◽  
Lawrence F Brass ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Electron Microscopy Analysis ◽  
Microscopy Analysis ◽  
Plug Formation

scholarly journals A 3D Cell Death Assay to Quantitatively Determine Ferroptosis in Spheroids

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 703 ◽  
Author(s):  
Robin Demuynck ◽  
Iuliia Efimova ◽  
Abraham Lin ◽  
Heidi Declercq ◽  
Dmitri V. Krysko
Keyword(s):  
Cell Death ◽  
Three Dimensional ◽  
Drug Efficacy ◽  
Cost Effective ◽  
3D Cultures ◽  
Cell Death Assay ◽  
Tumour Spheroids ◽  
A Cell ◽  
Triton X

The failure of drug efficacy in clinical trials remains a big issue in cancer research. This is largely due to the limitations of two-dimensional (2D) cell cultures, the most used tool in drug screening. Nowadays, three-dimensional (3D) cultures, including spheroids, are acknowledged to be a better model of the in vivo environment, but detailed cell death assays for 3D cultures (including those for ferroptosis) are scarce. In this work, we show that a new cell death analysis method, named 3D Cell Death Assay (3DELTA), can efficiently determine different cell death types including ferroptosis and quantitatively assess cell death in tumour spheroids. Our method uses Sytox dyes as a cell death marker and Triton X-100, which efficiently permeabilizes all cells in spheroids, was used to establish 100% cell death. After optimization of Sytox concentration, Triton X-100 concentration and timing, we showed that the 3DELTA method was able to detect signals from all cells without the need to disaggregate spheroids. Moreover, in this work we demonstrated that 2D experiments cannot be extrapolated to 3D cultures as 3D cultures are less sensitive to cell death induction. In conclusion, 3DELTA is a more cost-effective way to identify and measure cell death type in 3D cultures, including spheroids.

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