TY - JOUR
T1 - Quantitative Imaging of Labile Zn2+ in the Golgi Apparatus Using a Localizable Small-Molecule Fluorescent Probe
AU - Kowada, Toshiyuki
AU - Watanabe, Tomomi
AU - Amagai, Yuta
AU - Liu, Rong
AU - Yamada, Momo
AU - Takahashi, Hiroto
AU - Matsui, Toshitaka
AU - Inaba, Kenji
AU - Mizukami, Shin
N1 - Funding Information:
This work was supported by MEXT KAKENHI Grant Numbers JP18H04605 and JP19H05284 , by JSPS KAKENHI Grant Numbers JP15H03120 , JP17K05921 , JP18H02102 , JP18F18340 , JP19K22241 , and JP 20K05702 , by IMRAM projects (T.K. and Y.A.), and by the Suzuken Memorial Foundation , the Uehara Memorial Foundation , the Takeda Science Foundation , the Astellas Foundation for Research on Metabolic Disorders , the Tokyo Biochemical Research Foundation , the Kowa Life Science Foundation , the Nakatani Foundation , and the " Dynamic Alliance for Open Innovation Bridging Human, Environment and Materials " Research Program in the "Network Joint Research Center for Materials and Devices". We thank Tagen Central Analytical Facility for providing NMR and MS instruments and Mr. D. Unabara for his advice in qNMR measurement. We are also grateful to supercomputing resources at Cyberscience Center in Tohoku University for the use of the Gaussian program.
Funding Information:
This work was supported by MEXT KAKENHI Grant Numbers JP18H04605 and JP19H05284, by JSPS KAKENHI Grant Numbers JP15H03120, JP17K05921, JP18H02102, JP18F18340, JP19K22241, and JP20K05702, by IMRAM projects (T.K. and Y.A.), and by the Suzuken Memorial Foundation, the Uehara Memorial Foundation, the Takeda Science Foundation, the Astellas Foundation for Research on Metabolic Disorders, the Tokyo Biochemical Research Foundation, the Kowa Life Science Foundation, the Nakatani Foundation, and the “Dynamic Alliance for Open Innovation Bridging Human, Environment and Materials” Research Program in the "Network Joint Research Center for Materials and Devices". We thank Tagen Central Analytical Facility for providing NMR and MS instruments and Mr. D. Unabara for his advice in qNMR measurement. We are also grateful to supercomputing resources at Cyberscience Center in Tohoku University for the use of the Gaussian program. T.K. Y.A. K.I. and S.M. constructed the research strategy. T.K. T.W. and R.L. synthesized and characterized the fluorescent probe. T.K. T.W. Y.A. M.Y. H.T. and T.M. constructed the plasmids. H.T. prepared the recombinant MBP-HaloTag protein. T.K. T.W. and R.L. performed the cell imaging experiments. T.K. T.W. and S.M. wrote the initial draft of the manuscript, and Y.A. T.M. and K.I. joined to finalize the manuscript. The authors declare no competing interests.
Publisher Copyright:
© 2020 Elsevier Ltd
PY - 2020/12/17
Y1 - 2020/12/17
N2 - Fluorescent Zn2+ probes used for the quantitative analysis of labile Zn2+ concentration ([Zn2+]) in target organelles are crucial for understanding the role of Zn2+ in biological processes. Although several fluorescent Zn2+ probes have been developed to date, there is still a lack of consensus concerning the [Zn2+] in intracellular organelles. In this study, we describe the development of ZnDA-1H, a small-molecule fluorescent probe for Zn2+, which exhibits less pH sensitivity, high Zn2+ selectivity, and large fluorescence enhancement upon binding to Zn2+. Through protein labeling technology, ZnDA-1H was precisely targeted in various intracellular organelles, such as the nucleus, mitochondria, endoplasmic reticulum, and Golgi apparatus. ZnDA-1H exhibited a reversible fluorescence response toward labile Zn2+ in these organelles in live cells. Using this probe, the [Zn2+] in the Golgi apparatus was estimated to be 25 ± 1 nM, suggesting that labile Zn2+ plays a physiological role in the secretory pathway.
AB - Fluorescent Zn2+ probes used for the quantitative analysis of labile Zn2+ concentration ([Zn2+]) in target organelles are crucial for understanding the role of Zn2+ in biological processes. Although several fluorescent Zn2+ probes have been developed to date, there is still a lack of consensus concerning the [Zn2+] in intracellular organelles. In this study, we describe the development of ZnDA-1H, a small-molecule fluorescent probe for Zn2+, which exhibits less pH sensitivity, high Zn2+ selectivity, and large fluorescence enhancement upon binding to Zn2+. Through protein labeling technology, ZnDA-1H was precisely targeted in various intracellular organelles, such as the nucleus, mitochondria, endoplasmic reticulum, and Golgi apparatus. ZnDA-1H exhibited a reversible fluorescence response toward labile Zn2+ in these organelles in live cells. Using this probe, the [Zn2+] in the Golgi apparatus was estimated to be 25 ± 1 nM, suggesting that labile Zn2+ plays a physiological role in the secretory pathway.
KW - Golgi apparatus
KW - Zn
KW - fluorescence imaging
KW - quantitative analysis
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U2 - 10.1016/j.chembiol.2020.09.003
DO - 10.1016/j.chembiol.2020.09.003
M3 - Article
C2 - 32997976
AN - SCOPUS:85092670806
SN - 2451-9448
VL - 27
SP - 1521-1531.e8
JO - Cell Chemical Biology
JF - Cell Chemical Biology
IS - 12
ER -