Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism
Genetically encoded iNap sensors allow imaging of NADPH with high spatiotemporal resolution in living systems. The iNaps cover physiologically relevant NADPH concentrations and are demonstrated in mammalian cells and live zebrafish. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is esse...
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| Veröffentlicht in: | Nature methods 2017-07, Vol.14 (7), p.720-728 |
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| creator | Tao, Rongkun Zhao, Yuzheng Chu, Huanyu Wang, Aoxue Zhu, Jiahuan Chen, Xianjun Zou, Yejun Shi, Mei Liu, Renmei Su, Ni Du, Jiulin Zhou, Hai-Meng Zhu, Linyong Qian, Xuhong Liu, Haiyan Loscalzo, Joseph Yang, Yi |
| description | Genetically encoded iNap sensors allow imaging of NADPH with high spatiotemporal resolution in living systems. The iNaps cover physiologically relevant NADPH concentrations and are demonstrated in mammalian cells and live zebrafish.
Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is essential for biosynthetic reactions and antioxidant functions; however, detection of NADPH metabolism in living cells remains technically challenging. We develop and characterize ratiometric, pH-resistant, genetically encoded fluorescent indicators for NADPH (iNap sensors) with various affinities and wide dynamic range. iNap sensors enabled quantification of cytosolic and mitochondrial NADPH pools that are controlled by cytosolic NAD
+
kinase levels and revealed cellular NADPH dynamics under oxidative stress depending on glucose availability. We found that mammalian cells have a strong tendency to maintain physiological NADPH homeostasis, which is regulated by glucose-6-phosphate dehydrogenase and AMP kinase. Moreover, using the iNap sensors we monitor NADPH fluctuations during the activation of macrophage cells or wound response
in vivo
. These data demonstrate that the iNap sensors will be valuable tools for monitoring NADPH dynamics in live cells and gaining new insights into cell metabolism. |
| doi_str_mv | 10.1038/nmeth.4306 |
| format | Article |
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Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is essential for biosynthetic reactions and antioxidant functions; however, detection of NADPH metabolism in living cells remains technically challenging. We develop and characterize ratiometric, pH-resistant, genetically encoded fluorescent indicators for NADPH (iNap sensors) with various affinities and wide dynamic range. iNap sensors enabled quantification of cytosolic and mitochondrial NADPH pools that are controlled by cytosolic NAD
+
kinase levels and revealed cellular NADPH dynamics under oxidative stress depending on glucose availability. We found that mammalian cells have a strong tendency to maintain physiological NADPH homeostasis, which is regulated by glucose-6-phosphate dehydrogenase and AMP kinase. Moreover, using the iNap sensors we monitor NADPH fluctuations during the activation of macrophage cells or wound response
in vivo
. These data demonstrate that the iNap sensors will be valuable tools for monitoring NADPH dynamics in live cells and gaining new insights into cell metabolism.</description><identifier>ISSN: 1548-7091</identifier><identifier>EISSN: 1548-7105</identifier><identifier>DOI: 10.1038/nmeth.4306</identifier><identifier>PMID: 28581494</identifier><language>eng</language><publisher>New York: Nature Publishing Group US</publisher><subject>14/10 ; 14/35 ; 631/1647/1888/2249 ; 631/1647/245/2225 ; 631/61/338/469 ; 631/80/2373 ; AMP-Activated Protein Kinases - genetics ; AMP-Activated Protein Kinases - metabolism ; Animals ; Bioinformatics ; Biological Microscopy ; Biological Techniques ; Biomedical and Life Sciences ; Biomedical Engineering/Biotechnology ; Biosensors ; Cell activation ; Cell Survival ; Cells (biology) ; Coding ; Dynamic range ; Fluorescence ; Fluorescent indicators ; Fluorescent proteins ; Gene Expression Regulation - physiology ; Genetic code ; Genetics ; Glucose ; Glucose 6 phosphate dehydrogenase ; Glucose-6-phosphate ; Glucosephosphate dehydrogenase ; Homeostasis ; Humans ; Indicators ; Life Sciences ; Luminescent Proteins - chemistry ; Luminescent Proteins - genetics ; Luminescent Proteins - metabolism ; Macrophages ; Mammalian cells ; Mammals ; Metabolism ; Mice ; Mitochondria ; Models, Molecular ; NADP - metabolism ; NADPH-diaphorase ; Nicotinamide ; Nicotinamide adenine dinucleotide ; Nicotinamide adenine dinucleotide phosphate ; Oxidative Stress ; Properties ; Protein Binding ; Protein Conformation ; Protein Domains ; Protein Engineering ; Proteomics ; Sensors</subject><ispartof>Nature methods, 2017-07, Vol.14 (7), p.720-728</ispartof><rights>Springer Nature America, Inc. 2017</rights><rights>COPYRIGHT 2017 Nature Publishing Group</rights><rights>Copyright Nature Publishing Group Jul 2017</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c509t-9ac3f853f816151481ab990ca146dab01e5fdb6356debc8b9a5dfaf1a7519843</citedby><cites>FETCH-LOGICAL-c509t-9ac3f853f816151481ab990ca146dab01e5fdb6356debc8b9a5dfaf1a7519843</cites><orcidid>0000-0001-7896-1184</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1038/nmeth.4306$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1038/nmeth.4306$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>230,314,776,780,881,27903,27904,41467,42536,51297</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/28581494$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Tao, Rongkun</creatorcontrib><creatorcontrib>Zhao, Yuzheng</creatorcontrib><creatorcontrib>Chu, Huanyu</creatorcontrib><creatorcontrib>Wang, Aoxue</creatorcontrib><creatorcontrib>Zhu, Jiahuan</creatorcontrib><creatorcontrib>Chen, Xianjun</creatorcontrib><creatorcontrib>Zou, Yejun</creatorcontrib><creatorcontrib>Shi, Mei</creatorcontrib><creatorcontrib>Liu, Renmei</creatorcontrib><creatorcontrib>Su, Ni</creatorcontrib><creatorcontrib>Du, Jiulin</creatorcontrib><creatorcontrib>Zhou, Hai-Meng</creatorcontrib><creatorcontrib>Zhu, Linyong</creatorcontrib><creatorcontrib>Qian, Xuhong</creatorcontrib><creatorcontrib>Liu, Haiyan</creatorcontrib><creatorcontrib>Loscalzo, Joseph</creatorcontrib><creatorcontrib>Yang, Yi</creatorcontrib><title>Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism</title><title>Nature methods</title><addtitle>Nat Methods</addtitle><addtitle>Nat Methods</addtitle><description>Genetically encoded iNap sensors allow imaging of NADPH with high spatiotemporal resolution in living systems. The iNaps cover physiologically relevant NADPH concentrations and are demonstrated in mammalian cells and live zebrafish.
Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is essential for biosynthetic reactions and antioxidant functions; however, detection of NADPH metabolism in living cells remains technically challenging. We develop and characterize ratiometric, pH-resistant, genetically encoded fluorescent indicators for NADPH (iNap sensors) with various affinities and wide dynamic range. iNap sensors enabled quantification of cytosolic and mitochondrial NADPH pools that are controlled by cytosolic NAD
+
kinase levels and revealed cellular NADPH dynamics under oxidative stress depending on glucose availability. We found that mammalian cells have a strong tendency to maintain physiological NADPH homeostasis, which is regulated by glucose-6-phosphate dehydrogenase and AMP kinase. Moreover, using the iNap sensors we monitor NADPH fluctuations during the activation of macrophage cells or wound response
in vivo
. These data demonstrate that the iNap sensors will be valuable tools for monitoring NADPH dynamics in live cells and gaining new insights into cell metabolism.</description><subject>14/10</subject><subject>14/35</subject><subject>631/1647/1888/2249</subject><subject>631/1647/245/2225</subject><subject>631/61/338/469</subject><subject>631/80/2373</subject><subject>AMP-Activated Protein Kinases - genetics</subject><subject>AMP-Activated Protein Kinases - metabolism</subject><subject>Animals</subject><subject>Bioinformatics</subject><subject>Biological Microscopy</subject><subject>Biological Techniques</subject><subject>Biomedical and Life Sciences</subject><subject>Biomedical Engineering/Biotechnology</subject><subject>Biosensors</subject><subject>Cell activation</subject><subject>Cell Survival</subject><subject>Cells (biology)</subject><subject>Coding</subject><subject>Dynamic range</subject><subject>Fluorescence</subject><subject>Fluorescent indicators</subject><subject>Fluorescent proteins</subject><subject>Gene Expression Regulation - physiology</subject><subject>Genetic code</subject><subject>Genetics</subject><subject>Glucose</subject><subject>Glucose 6 phosphate dehydrogenase</subject><subject>Glucose-6-phosphate</subject><subject>Glucosephosphate dehydrogenase</subject><subject>Homeostasis</subject><subject>Humans</subject><subject>Indicators</subject><subject>Life Sciences</subject><subject>Luminescent Proteins - chemistry</subject><subject>Luminescent Proteins - genetics</subject><subject>Luminescent Proteins - metabolism</subject><subject>Macrophages</subject><subject>Mammalian cells</subject><subject>Mammals</subject><subject>Metabolism</subject><subject>Mice</subject><subject>Mitochondria</subject><subject>Models, Molecular</subject><subject>NADP - metabolism</subject><subject>NADPH-diaphorase</subject><subject>Nicotinamide</subject><subject>Nicotinamide adenine dinucleotide</subject><subject>Nicotinamide adenine dinucleotide phosphate</subject><subject>Oxidative Stress</subject><subject>Properties</subject><subject>Protein Binding</subject><subject>Protein Conformation</subject><subject>Protein Domains</subject><subject>Protein Engineering</subject><subject>Proteomics</subject><subject>Sensors</subject><issn>1548-7091</issn><issn>1548-7105</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2017</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNptkt9vFCEQx4nR2Fp98Q8wm_hiNHcyB-zCi8mlamvSqA_1mbDscKVhocJuk_vv5by2tiqE8GM-84UZhpCXQJdAmXwfR5wulpzR9hE5BMHlogMqHt-uqYID8qyUS0oZ4yvxlByspJDAFT8kP04w4uStCWHbYLRpwKFxYU4Zi8U4NQVjSbk0Ga_RhGbYRjN6W7ebOZjJp9gk13xdf_x-2tRnmD4FX8bn5IkzoeCLm_mInH_-dH58ujj7dvLleH22sIKqaaGMZU6KOqAFAVyC6ZWi1gBvB9NTQOGGvmWiHbC3sldGDM44MJ0AJTk7Ih_2sldzP-Kwe282QV9lP5q81cl4_dAS_YXepGstauN0VQXe3Ajk9HPGMunR17BDMBHTXDQo2gKTkrGKvv4LvUxzjjW6SkGnWq4o_0NtTEDto0v1XrsT1WuuOgaqo22llv-hah-w5jZFdL6eP3B4u3ewOZWS0d3FCFTvakD_rgG9q4EKv7qflTv09tMr8G4PlGqKG8z3QvlX7he_b7xK</recordid><startdate>20170701</startdate><enddate>20170701</enddate><creator>Tao, 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encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism</title><author>Tao, Rongkun ; Zhao, Yuzheng ; Chu, Huanyu ; Wang, Aoxue ; Zhu, Jiahuan ; Chen, Xianjun ; Zou, Yejun ; Shi, Mei ; Liu, Renmei ; Su, Ni ; Du, Jiulin ; Zhou, Hai-Meng ; Zhu, Linyong ; Qian, Xuhong ; Liu, Haiyan ; Loscalzo, Joseph ; Yang, Yi</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c509t-9ac3f853f816151481ab990ca146dab01e5fdb6356debc8b9a5dfaf1a7519843</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2017</creationdate><topic>14/10</topic><topic>14/35</topic><topic>631/1647/1888/2249</topic><topic>631/1647/245/2225</topic><topic>631/61/338/469</topic><topic>631/80/2373</topic><topic>AMP-Activated Protein Kinases - genetics</topic><topic>AMP-Activated Protein Kinases - metabolism</topic><topic>Animals</topic><topic>Bioinformatics</topic><topic>Biological Microscopy</topic><topic>Biological 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cells</topic><topic>Mammals</topic><topic>Metabolism</topic><topic>Mice</topic><topic>Mitochondria</topic><topic>Models, Molecular</topic><topic>NADP - metabolism</topic><topic>NADPH-diaphorase</topic><topic>Nicotinamide</topic><topic>Nicotinamide adenine dinucleotide</topic><topic>Nicotinamide adenine dinucleotide phosphate</topic><topic>Oxidative Stress</topic><topic>Properties</topic><topic>Protein Binding</topic><topic>Protein Conformation</topic><topic>Protein Domains</topic><topic>Protein Engineering</topic><topic>Proteomics</topic><topic>Sensors</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Tao, Rongkun</creatorcontrib><creatorcontrib>Zhao, Yuzheng</creatorcontrib><creatorcontrib>Chu, Huanyu</creatorcontrib><creatorcontrib>Wang, Aoxue</creatorcontrib><creatorcontrib>Zhu, Jiahuan</creatorcontrib><creatorcontrib>Chen, Xianjun</creatorcontrib><creatorcontrib>Zou, Yejun</creatorcontrib><creatorcontrib>Shi, 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Xuhong</au><au>Liu, Haiyan</au><au>Loscalzo, Joseph</au><au>Yang, Yi</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism</atitle><jtitle>Nature methods</jtitle><stitle>Nat Methods</stitle><addtitle>Nat Methods</addtitle><date>2017-07-01</date><risdate>2017</risdate><volume>14</volume><issue>7</issue><spage>720</spage><epage>728</epage><pages>720-728</pages><issn>1548-7091</issn><eissn>1548-7105</eissn><abstract>Genetically encoded iNap sensors allow imaging of NADPH with high spatiotemporal resolution in living systems. The iNaps cover physiologically relevant NADPH concentrations and are demonstrated in mammalian cells and live zebrafish.
Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is essential for biosynthetic reactions and antioxidant functions; however, detection of NADPH metabolism in living cells remains technically challenging. We develop and characterize ratiometric, pH-resistant, genetically encoded fluorescent indicators for NADPH (iNap sensors) with various affinities and wide dynamic range. iNap sensors enabled quantification of cytosolic and mitochondrial NADPH pools that are controlled by cytosolic NAD
+
kinase levels and revealed cellular NADPH dynamics under oxidative stress depending on glucose availability. We found that mammalian cells have a strong tendency to maintain physiological NADPH homeostasis, which is regulated by glucose-6-phosphate dehydrogenase and AMP kinase. Moreover, using the iNap sensors we monitor NADPH fluctuations during the activation of macrophage cells or wound response
in vivo
. These data demonstrate that the iNap sensors will be valuable tools for monitoring NADPH dynamics in live cells and gaining new insights into cell metabolism.</abstract><cop>New York</cop><pub>Nature Publishing Group US</pub><pmid>28581494</pmid><doi>10.1038/nmeth.4306</doi><tpages>9</tpages><orcidid>https://orcid.org/0000-0001-7896-1184</orcidid><oa>free_for_read</oa></addata></record> |
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| ispartof | Nature methods, 2017-07, Vol.14 (7), p.720-728 |
| issn | 1548-7091 1548-7105 |
| language | eng |
| recordid | cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_5555402 |
| source | MEDLINE; SpringerLink Journals; Nature |
| subjects | 14/10 14/35 631/1647/1888/2249 631/1647/245/2225 631/61/338/469 631/80/2373 AMP-Activated Protein Kinases - genetics AMP-Activated Protein Kinases - metabolism Animals Bioinformatics Biological Microscopy Biological Techniques Biomedical and Life Sciences Biomedical Engineering/Biotechnology Biosensors Cell activation Cell Survival Cells (biology) Coding Dynamic range Fluorescence Fluorescent indicators Fluorescent proteins Gene Expression Regulation - physiology Genetic code Genetics Glucose Glucose 6 phosphate dehydrogenase Glucose-6-phosphate Glucosephosphate dehydrogenase Homeostasis Humans Indicators Life Sciences Luminescent Proteins - chemistry Luminescent Proteins - genetics Luminescent Proteins - metabolism Macrophages Mammalian cells Mammals Metabolism Mice Mitochondria Models, Molecular NADP - metabolism NADPH-diaphorase Nicotinamide Nicotinamide adenine dinucleotide Nicotinamide adenine dinucleotide phosphate Oxidative Stress Properties Protein Binding Protein Conformation Protein Domains Protein Engineering Proteomics Sensors |
| title | Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-26T09%3A38%3A11IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_pubme&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Genetically%20encoded%20fluorescent%20sensors%20reveal%20dynamic%20regulation%20of%20NADPH%20metabolism&rft.jtitle=Nature%20methods&rft.au=Tao,%20Rongkun&rft.date=2017-07-01&rft.volume=14&rft.issue=7&rft.spage=720&rft.epage=728&rft.pages=720-728&rft.issn=1548-7091&rft.eissn=1548-7105&rft_id=info:doi/10.1038/nmeth.4306&rft_dat=%3Cgale_pubme%3EA497319706%3C/gale_pubme%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1917964904&rft_id=info:pmid/28581494&rft_galeid=A497319706&rfr_iscdi=true |