Rational design of a monomeric and photostable far‐red fluorescent protein for fluorescence imaging in vivo |
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| Authors: | William Clay Gustafson Rubén Ruiz‐González Luca Signor Fanny Marzocca Franck Borel Matthew P. Klassen Kalpana Makhijani Antoine Royant Yuh‐Nung Jan William A. Weiss Su Guo Xiaokun Shu |
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| Affiliation: | 1. Department of Pediatrics, Departments of Neurology and Neurological Surgery, University of California – San Francisco, San Francisco, California;2. Institut Químic De Sarrià, Universitat Ramon Llull, Barcelona, Spain;3. Institut De Biologie Structurale (IBS), University of Grenoble Alpes, CNRS, CEA, Grenoble, France;4. Howard Hughes Medical Institute, Department of Physiology, University of California – San Francisco, San Francisco, California;5. Department of Pharmaceutical Chemistry, Cardiovascular Research Institute, University of California – San Francisco, San Francisco, California;6. European Synchrotron Radiation Facility, Grenoble, France;7. Helen Diller Family Comprehensive Cancer Center, University of California – San Francisco, San Francisco, California;8. Department of Bioengineering and Therapeutic Science, Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Institute of Human Genetics, University of California, San Francisco, California |
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| Abstract: | Fluorescent proteins (FPs) are powerful tools for cell and molecular biology. Here based on structural analysis, a blue‐shifted mutant of a recently engineered monomeric infrared fluorescent protein (mIFP) has been rationally designed. This variant, named iBlueberry, bears a single mutation that shifts both excitation and emission spectra by approximately 40 nm. Furthermore, iBlueberry is four times more photostable than mIFP, rendering it more advantageous for imaging protein dynamics. By tagging iBlueberry to centrin, it has been demonstrated that the fusion protein labels the centrosome in the developing zebrafish embryo. Together with GFP‐labeled nucleus and tdTomato‐labeled plasma membrane, time‐lapse imaging to visualize the dynamics of centrosomes in radial glia neural progenitors in the intact zebrafish brain has been demonstrated. It is further shown that iBlueberry can be used together with mIFP in two‐color protein labeling in living cells and in two‐color tumor labeling in mice. |
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| Keywords: | rational design fluorescent proteins fluorescence imaging bacterial phytochrome |
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