Toll-like receptor–independent gene induction program activated by mammalian DNA escaped from apoptotic DNA degradation

Deoxyribonuclease (DNase) II in macrophages cleaves the DNA of engulfed apoptotic cells and of nuclei expelled from erythroid precursor cells. DNase II–deficient mouse embryos accumulate undigested DNA in macrophages, and die in feto because of the activation of the interferon β (IFNβ) gene. Here, we found that the F4/80-positive macrophages in DNase II−/− fetal liver specifically produce a set of cytokines such as IFNβ, TNFα, and CXCL10. Whereas, IFN-inducible genes (2′5′-oligo(A) synthetase, IRF7, and ISG15) were expressed not only in macrophages but also in other F4/80-negative cells. When DNase II−/− macrophages or embryonal fibroblasts engulfed apoptotic cells, they expressed the IFNβ and CXCL10 genes. The ablation of Toll-like receptor (TLR) 3 and 9, or their adaptor molecules (MyD88 and TRIF), had no effect on the lethality of the DNase II−/− mice. These results indicate that there is a TLR-independent sensing mechanism to activate the innate immunity for the endogenous DNA escaping lysosomal degradation

Role of PERK in mitochondrial function

Mitochondria play a central role in the function of brown adipocytes (BAs). Although mitochondrial biogenesis, which is indispensable for thermogenesis, is regulated by coordination between nuclear DNA transcription and mitochondrial DNA transcription, the molecular mechanisms of mitochondrial development during BA differentiation are largely unknown. Here, we show the importance of the ER-resident sensor PKR-like ER kinase (PERK) in the mitochondrial thermogenesis of brown adipose tissue. During BA differentiation, PERK is physiologically phosphorylated independently of the ER stress. This PERK phosphorylation induces transcriptional activation by GA-binding protein transcription factor α subunit (GABPα), which is required for mitochondrial inner membrane protein biogenesis, and this novel role of PERK is involved in maintaining the body temperatures of mice during cold exposure. Our findings demonstrate that mitochondrial development regulated by the PERK–GABPα axis is indispensable for thermogenesis in brown adipose tissue

Real time monitoring of endogenous cytoplasmic mRNA using linear antisense 2′-O-methyl RNA probes in living cells

Visualization and monitoring of endogenous mRNA in the cytoplasm of living cells promises a significant comprehension of refined post-transcriptional regulation. Fluorescently labeled linear antisense oligonucleotides can bind to natural mRNA in a sequence-specific way and, therefore, provide a powerful tool in probing endogenous mRNA. Here, we investigated the feasibility of using linear antisense probes to monitor the variable and dynamic expression of endogenous cytoplasmic mRNAs. Two linear antisense 2′-O-methyl RNA probes, which have different interactive fluorophores at the 5′-end of one probe and at the 3′-end of the other, were used to allow fluorescence resonance energy transfer (FRET) upon hybridization to the target mRNA. By characterizing the formation of the probe-mRNA hybrids in living cells, we found that the probe composition and concentration are crucial parameters in the visualization of endogenous mRNA with high specificity. Furthermore, rapid hybridization (within 1 min) of the linear antisense probe enabled us to visualize dynamic processes of endogenous c-fos mRNA, such as fast elevation of levels after gene induction and the localization of c-fos mRNA in stress granules in response to cellular stress. Thus, our approach provides a basis for real time monitoring of endogenous cytoplasmic mRNA in living cells

Measurement of temperature distribution inside X-ray irradiated cells

It is becoming recognized that cellular temperature depends on the intracellular condition. The heat balance of chemical reactions inside various organelles may change significantly due to external stress, such as ionizing irradiation. In the present study, HeLa cells were investigated a fluorescent chemical probe, Cellular Thermoprobe for Fluorescence Ratio, to determine temperature of each cell after X-ray irradiation of 10 Gy. We used two methods to irradiate the cells with X-rays. The first is to irradiate the entire dish with X-rays. The second method is to limit the irradiation range and irradiate X-rays separately in the irradiated area and the non-irradiated area. After irradiation with X-rays, it was observed by the microscope. The intensity and distribution of the two fluorescences were analyzed using an image analysis software, ImageJ. The temperatures were then determined from the obtained fluorescent ratios based on a calibration curve. The intracellular temperature were significantly different even between cells in same dish. For the irradiated cells, the temperature was more dispersed among the cells and prone to be statically higher than that of the control cells. Fine temperature-distribution over the cell including various organelles will be also reported.日本放射線影響学会第64回大

Cell-autonomous control of intracellular temperature by unsaturation of phospholipid acyl chains

【研究成果】寒くなると細胞は自ら温度を上げようとする --細胞における新たな温度調節機構の発見--. 京都大学プレスリリース. 2022-03-16.Intracellular temperature affects a wide range of cellular functions in living organisms. However, it remains unclear whether temperature in individual animal cells is controlled autonomously as a response to fluctuations in environmental temperature. Using two distinct intracellular thermometers, we find that the intracellular temperature of steady-state Drosophila S2 cells is maintained in a manner dependent on Δ9-fatty acid desaturase DESAT1, which introduces a double bond at the Δ9 position of the acyl moiety of acyl-CoA. The DESAT1-mediated increase of intracellular temperature is caused by the enhancement of F₁F₀-ATPase-dependent mitochondrial respiration, which is coupled with thermogenesis. We also reveal that F₁F₀-ATPase-dependent mitochondrial respiration is potentiated by cold exposure through the remodeling of mitochondrial cristae structures via DESAT1-dependent unsaturation of mitochondrial phospholipid acyl chains. Based on these findings, we propose a cell-autonomous mechanism for intracellular temperature control during environmental temperature changes