Probing Subcellular Iron Availability with Genetically Encoded Nitric Oxide Biosensors
Cellular iron supply is required for various biochemical processes. Measuring bioavailable iron in cells aids in obtaining a better understanding of its biochemical activities but is technically challenging. Existing techniques have several constraints that make precise localization difficult, and t...
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| Format: | Article |
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MDPI AG
2022-10-01
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| Series: | Biosensors |
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| Online Access: | https://www.mdpi.com/2079-6374/12/10/903 |
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| author | Gulsah Sevimli Amy E. Alston Felix Funk Beat Flühmann Roland Malli Wolfgang F. Graier Emrah Eroglu |
| author_facet | Gulsah Sevimli Amy E. Alston Felix Funk Beat Flühmann Roland Malli Wolfgang F. Graier Emrah Eroglu |
| author_sort | Gulsah Sevimli |
| collection | DOAJ |
| description | Cellular iron supply is required for various biochemical processes. Measuring bioavailable iron in cells aids in obtaining a better understanding of its biochemical activities but is technically challenging. Existing techniques have several constraints that make precise localization difficult, and the lack of a functional readout makes it unclear whether the tested labile iron is available for metalloproteins. Here, we use geNOps; a ferrous iron-dependent genetically encoded fluorescent nitric oxide (NO) biosensor, to measure available iron in cellular locales. We exploited the nitrosylation-dependent fluorescence quenching of geNOps as a direct readout for cellular iron absorption, distribution, and availability. Our findings show that, in addition to ferrous iron salts, the complex of iron (III) with N,N’-bis (2-hydroxybenzyl)ethylenediamine-N,N’-diacetic acid (HBED) can activate the iron (II)-dependent NO probe within intact cells. Cell treatment for only 20 min with iron sucrose was also sufficient to activate the biosensor in the cytosol and mitochondria significantly; however, ferric carboxymaltose failed to functionalize the probe, even after 2 h of cell treatment. Our findings show that the geNOps approach detects available iron (II) in cultured cells and can be applied to assay functional iron (II) at the (sub)cellular level. |
| first_indexed | 2024-03-09T20:34:47Z |
| format | Article |
| id | doaj.art-31184a3dafdc4f20a605c5aaad6a06c6 |
| institution | Directory Open Access Journal |
| issn | 2079-6374 |
| language | English |
| last_indexed | 2024-03-09T20:34:47Z |
| publishDate | 2022-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biosensors |
| spelling | doaj.art-31184a3dafdc4f20a605c5aaad6a06c62023-11-23T23:12:31ZengMDPI AGBiosensors2079-63742022-10-01121090310.3390/bios12100903Probing Subcellular Iron Availability with Genetically Encoded Nitric Oxide BiosensorsGulsah Sevimli0Amy E. Alston1Felix Funk2Beat Flühmann3Roland Malli4Wolfgang F. Graier5Emrah Eroglu6Molecular Biology, Genetics and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul 34956, TurkeyCSLVifor Ltd., Redwood City, CA 94063, USACSL Vifor Ltd., Flughofstrasse 61, CH-8152 Glattbrugg, SwitzerlandCSL Vifor Ltd., Flughofstrasse 61, CH-8152 Glattbrugg, SwitzerlandDepartment of Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, 8010 Graz, AustriaDepartment of Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, 8010 Graz, AustriaMolecular Biology, Genetics and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul 34956, TurkeyCellular iron supply is required for various biochemical processes. Measuring bioavailable iron in cells aids in obtaining a better understanding of its biochemical activities but is technically challenging. Existing techniques have several constraints that make precise localization difficult, and the lack of a functional readout makes it unclear whether the tested labile iron is available for metalloproteins. Here, we use geNOps; a ferrous iron-dependent genetically encoded fluorescent nitric oxide (NO) biosensor, to measure available iron in cellular locales. We exploited the nitrosylation-dependent fluorescence quenching of geNOps as a direct readout for cellular iron absorption, distribution, and availability. Our findings show that, in addition to ferrous iron salts, the complex of iron (III) with N,N’-bis (2-hydroxybenzyl)ethylenediamine-N,N’-diacetic acid (HBED) can activate the iron (II)-dependent NO probe within intact cells. Cell treatment for only 20 min with iron sucrose was also sufficient to activate the biosensor in the cytosol and mitochondria significantly; however, ferric carboxymaltose failed to functionalize the probe, even after 2 h of cell treatment. Our findings show that the geNOps approach detects available iron (II) in cultured cells and can be applied to assay functional iron (II) at the (sub)cellular level.https://www.mdpi.com/2079-6374/12/10/903labile ironcellular iron uptakefluorescent biosensorgeNOpsFerinjectVenofer |
| spellingShingle | Gulsah Sevimli Amy E. Alston Felix Funk Beat Flühmann Roland Malli Wolfgang F. Graier Emrah Eroglu Probing Subcellular Iron Availability with Genetically Encoded Nitric Oxide Biosensors Biosensors labile iron cellular iron uptake fluorescent biosensor geNOps Ferinject Venofer |
| title | Probing Subcellular Iron Availability with Genetically Encoded Nitric Oxide Biosensors |
| title_full | Probing Subcellular Iron Availability with Genetically Encoded Nitric Oxide Biosensors |
| title_fullStr | Probing Subcellular Iron Availability with Genetically Encoded Nitric Oxide Biosensors |
| title_full_unstemmed | Probing Subcellular Iron Availability with Genetically Encoded Nitric Oxide Biosensors |
| title_short | Probing Subcellular Iron Availability with Genetically Encoded Nitric Oxide Biosensors |
| title_sort | probing subcellular iron availability with genetically encoded nitric oxide biosensors |
| topic | labile iron cellular iron uptake fluorescent biosensor geNOps Ferinject Venofer |
| url | https://www.mdpi.com/2079-6374/12/10/903 |
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