Biliverdin Reductase-A Deficiency Brighten and Sensitize Biliverdin-binding Chromoproteins

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  • Kobachi Kenju
    Laboratory of Bioimaging and Cell Signaling, Research Center for Dynamic Living Systems, Graduate School of Biostudies, Kyoto University
  • Kuno Sota
    Department of Molecular and Cellular Physiology, Graduate School of Medicine, Kyoto University
  • Sato Shinya
    Laboratory of Bioimaging and Cell Signaling, Research Center for Dynamic Living Systems, Graduate School of Biostudies, Kyoto University
  • Sumiyama Kenta
    Laboratory for Mouse Genetic Engineering, RIKEN Center for Biosystems Dynamics Research
  • Matsuda Michiyuki
    Laboratory of Bioimaging and Cell Signaling, Research Center for Dynamic Living Systems, Graduate School of Biostudies, Kyoto University Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University
  • Terai Kenta
    Laboratory of Bioimaging and Cell Signaling, Research Center for Dynamic Living Systems, Graduate School of Biostudies, Kyoto University

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Abstract

<p>Tissue absorbance, light scattering, and autofluorescence are significantly lower in the near-infrared (NIR) range than in the visible range. Because of these advantages, NIR fluorescent proteins (FPs) are in high demand for in vivo imaging. Nevertheless, application of NIR FPs such as iRFP is still limited due to their dimness in mammalian cells. In contrast to GFP and its variants, iRFP requires biliverdin (BV) as a chromophore. The dimness of iRFP is at least partly due to rapid reduction of BV by biliverdin reductase-A (BLVRA). Here, we established biliverdin reductase-a knockout (Blvra–/–) mice to increase the intracellular BV concentration and, thereby, to enhance iRFP fluorescence intensity. As anticipated, iRFP fluorescence intensity was significantly increased in all examined tissues of Blvra–/– mice. Similarly, the genetically encoded calcium indicator NIR-GECO1, which is engineered based on another NIR FP, mIFP, exhibited a marked increase in fluorescence intensity in mouse embryonic fibroblasts derived from Blvra–/– mice. We expanded this approach to an NIR light-sensing optogenetic tool, the BphP1-PpsR2 system, which also requires BV as a chromophore. Again, deletion of the Blvra gene markedly enhanced the light response in HeLa cells. These results indicate that the Blvra–/– mouse is a versatile tool for the in vivo application of NIR FPs and NIR light-sensing optogenetic tools.</p><p>Key words: in vivo imaging, near-infrared fluorescent protein, biliverdin, biliverdin reductase, optogenetic tool</p>

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