FRETbased Biosensor Visualizes Execution Of Necroptosis In Vivo - Techno Pedia

Generation of SMART-Tg mice is a representation of the structure of the SMART biosensor ( above ) and its activation mechanism (below). SMART consists of the N-terminal Ypet, the helical KL domain modifications α1 and α4 of MLKL and the C-terminal of ECFP. Upon induction of necroptosis, activated and phosphorylated RIPK3 (pRIPK3) phosphorylates MLKL, resulting in the formation of MLKL oligomers. Thus, MLKL oligomers induce conformational changes in SMART, possibly through interactions, thus increasing FRET efficiency. Pseudokinase KL domain, Ypet-modified yellow fluorescent protein, ECFP-enhanced cyan fluorescent protein, RIPK3 phosphorylated pRIPK3. b Western blot probed with anti-GFP antibody showing SMART biosensor expression in various mouse tissues. Tissue extracts were prepared from the indicated organs from 8-week-old wild-type or SMART-Tg mice. The results are representative of two independent experiments. c Mice received intraperitoneal injection of thioglycolate, then peritoneal cells were harvested by rinsing the peritoneal cavity with cold PBS on day 4 post-injection. Isolated cells were stained with the indicated antibodies and analyzed by flow cytometry. The percentage of CD11b ⁺ F4/80 ⁺ cells represents the macrophage fraction; The YFP levels detected in this cell population are indicative of SMART expression. Results are representative of three independent experiments. WT wild type. d Peritoneal macrophages from SMART-Tg mice untreated or stimulated with BV6 (1 μM) + zVAD (20 μM) or BV6 (1 μM) + zVAD (20 μM) + GSK'872 (5 μM) for the indicated durations. Cell death was assessed using the LDH release assay. The results are the mean ± SD of triplicate samples and are representative of five independent experiments. e , f SMART-Tg mouse-derived peritoneal macrophages were stimulated as described in d and the FRET/CFP ratio was calculated. Pseudo-colored images show cellular changes in FRET/CFP ratio values ​​in response to the stimuli shown ( e ). The FRET/CFP response is color coded (right). White arrows indicate cells undergoing necroptosis. Scale bar, 20 μm. The maximum change is observed in the FRET/CFP ratio ( f ). Results are mean ± SE ( n = 11 cells per condition). Each dot represents one cell. The results are representative of four independent experiments. Statistical significance was determined by Dunnett's multiple comparison test ( d ) or by one-way ANOVA using Turkey's multiple comparison test ( f ). Credits: Communication Biology (2022). DOI: 10.1038/s42003-022-04300-0

Necroptosis is a regulated cell death (RCD) similar to apoptosis, the most studied type of RCD. In contrast to apoptosis, plasma membrane degradation occurs in the early stages of necroptotic cells. Because of this, scientists believe that necroptosis causes severe inflammation of the surrounding tissue and plays a role in inflammation-related diseases. However, it is unclear where and when necroptosis occurs under physiological and pathological conditions in vivo.

To overcome this problem, Prof.'s group. Nakano previously developed a biosensor for necroptosis called SMART (FRET-based sensor for MLKL activation by RIPK3). This biosensor is based on energy transfer resonance fluorescence (FRET). In a Nature Communications article published in 2018, they managed to characterize necroptosis in vitro using this FRET biosensor.

Now they have gone even further; developed transgenic mice with the SMART FRET biosensor for in vivo monitoring of necroptosis. "The aim of our project was to see when and where necroptosis occurs in vivo and understand its role in the disease context," said Dr. Murai, lead author of the study.

In their experiments, they first confirmed that necroptosis can be controlled in primary macrophages derived from SMART-Tg mice or in embryonic fibroblast clumps. They then implemented a cisplatin-induced acute kidney injury model in SMART-Tg mice.

“After much trial and error, we were finally able to monitor the execution of necroptosis in proximal tubular epithelial cells in SMART-Tg mice injected with cisplatin. Two-photon microscopy is essential for the effective monitoring of cell death in vivo," said Prof Nakano, lead author of the study.

"We believe that SMART Tg mice are a promising tool for visualizing necroptosis in vivo and helping us better understand the role of necroptosis, a relatively new form of cell death, in disease pathophysiology."

The findings are published in the journal Communications Biology .

Further information: Shin Murai et al., Generation of transgenic mice expressing the FRET biosensor, SMART, that responds to necroptosis, Communications Biology (2022). DOI: 10.1038/s42003-022-04300-0

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Excerpt : FRET-based biosensor visualizes the performance of necroptosis in vivo (9 December 2022) Accessed 17 December 2022 https://phys.org/news/2022-12-fret-based-biosensor-visualizes-necroptosis-alive.html- from

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