FRETbased Biosensor Visualizes Execution Of Necroptosis In Vivo

generation of SMART Tg mice. Representation of the SMART biosensor structure ( top ) and its activation mechanism (bottom). SMART consists of an N-terminal Ypet, a modified α1 and α4 helix of the KL domain of MLKL, and a C-terminal ECFP. During necroptosis induction, activated and phosphorylated RIPK3 (pRIPK3) phosphorylates MLKL, resulting in the formation of MLKL oligomers. MLKL oligomers then induce conformational changes in SMART, possibly through interactions, resulting in increased FRET efficiency. KL pseudokinase domain, Ypet-enhanced yellow fluorescent protein, ECFP-enhanced blue-green fluorescent protein, RIPK3 phosphorylates pRIPK3. b Western blots probed with anti-GFP antibodies show SMART biosensor expression in different tissues. Tissue extracts were prepared from the indicated organs of 8-week-old wild-type or SMART-Tg mice. The results are representative of two independent experiments. c Mice were injected intraperitoneally with thioglycolate and then peritoneal cells were harvested by washing the peritoneal cavity with ice-cold PBS on day 4 after injection. The isolated cells were stained with the indicated antibodies and analyzed by flow cytometry. The percentage of CD11b ⁺ F4/80 ⁺ cells represents the macrophage fraction; YFP levels detected in this cell population are indicative of SMART expression. The results are representative of three independent experiments. WT is wild type. d Peritoneal macrophages of SMART-Tg mice were not treated or stimulated with BV6 (1 μM) + zVAD (20 μM) or BV6 (1 μM) + zVAD (20 μM) + GSK'872 (5 μM) for the times indicated. Cell death was evaluated by an LDH release assay. Results are means ± SD of triplicate samples and are representative of five independent experiments. e , f Peritoneal macrophages of SMART-Tg mice were stimulated as described in d and FRET/CFP ratios were calculated. False-color images show cellular changes in FRET/CFP ratio values ​​in response to the indicated stimuli ( e ). FRET/CFP responses are coded according to the color scale (right). White arrowheads indicate cells undergoing necroptosis. Scale bar, 20 μm. Maximum change detected in the FRET/CFP ratio ( f ). Results are means ± SE ( n = 11 cells per condition). Each dot represents a cell. The results are representative of four independent experiments. Statistical significance was determined by two-way ANOVA using Dunnett's multiple comparison test ( d ) or one-way ANOVA using Turkey's multiple comparison test ( f ). Credit : Communication Biology (2022). DOI: 10.1038/s42003-022-04300-0

Necroptosis is apoptosis-like controlled cell death (RCD), the most studied type of RCD. In contrast to apoptosis, necroptosis occurs in cells with destruction of the plasma membrane. For this reason, scientists believe that necroptosis causes significant inflammation in surrounding tissue and plays a role in inflammatory disease. However, where and when necroptosis occurs under physiological and pathological conditions in vivo is not well understood.

Professor Dr. to solve this problem. Nakano's group developed a biosensor for necroptosis called SMART (FRET-based MLCL activation sensor of RIPK3). This biosensor is based on fluorescence resonance energy transfer (FRET). In a 2018 Nature Communications article, they successfully identified necrosis in vitro using this FRET biosensor.

This time they have gone one step further. They developed intelligent transgenic mice with a FRET biosensor to monitor necroptosis in vivo. "The goal of our project is to study when and where necroptosis occurs in vivo and to understand its role in the pathological context," said Dr. Murray, lead author of the study.

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In their experiments, they first confirmed that necroptosis can be regulated in primary macrophages or dead embryonic fibroblasts derived from SMART-Tg mice. They then applied a model of cisplatin-induced acute kidney injury to SMART-Tg mice.

"After much trial and error, we were finally able to monitor necroptosis function in proximal tubular epithelial cells of SMART-Tg mice injected with cisplatin time, which effectively kills cells important in death control, using the two." alive," said the study's lead author, Professor Nakano.

“We believe that intelligent TG mice are a promising tool for imaging necrosis in vivo and will help to better understand the role of this newly discovered form of cell death, necroptosis, in disease pathophysiology.”

These findings have been published in the journal Communications Biology .

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For more information: Shin Murai et al., Generation of Transgenic Mice Expressing a FRET Biosensor, Smart, Responsive to Necroptosis, Communication Biology (2022). DOI: 10.1038/s42003-022-04300-0

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Citation : FRET-based biosensor visual necroptosis in vivo (December 9, 2022) Retrieved December 15, 2022 from https://phys.org/news/2022-12-fret-based-biosensor-visualizes-necroptosis-vivo.html

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