Intracellular Ionic Strength Sensing Using NanoLuc
<div><p>Intracellular ionic strength regulates myriad cellular processes that are fundamental to cellular survival and proliferation, including protein activity, aggregation, phase separation, and cell volume. It could be altered by changes in the activity of cellular signaling pathways,...
محفوظ في:
| المؤلف الرئيسي: | |
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| مؤلفون آخرون: | , , |
| منشور في: |
2021
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| الموضوعات: | |
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| _version_ | 1864513515343052800 |
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| author | Tausif Altamash (3846304) |
| author2 | Wesam Ahmed (4870942) Saad Rasool (9764093) Kabir H. Biswas (5705864) |
| author2_role | author author author |
| author_facet | Tausif Altamash (3846304) Wesam Ahmed (4870942) Saad Rasool (9764093) Kabir H. Biswas (5705864) |
| author_role | author |
| dc.creator.none.fl_str_mv | Tausif Altamash (3846304) Wesam Ahmed (4870942) Saad Rasool (9764093) Kabir H. Biswas (5705864) |
| dc.date.none.fl_str_mv | 2021-01-12T03:00:00Z |
| dc.identifier.none.fl_str_mv | 10.3390/ijms22020677 |
| dc.relation.none.fl_str_mv | https://figshare.com/articles/journal_contribution/Intracellular_Ionic_Strength_Sensing_Using_NanoLuc/25867678 |
| dc.rights.none.fl_str_mv | CC BY 4.0 info:eu-repo/semantics/openAccess |
| dc.subject.none.fl_str_mv | Biological sciences Biochemistry and cell biology bioluminescence biosensor ionic strength NanoLuc |
| dc.title.none.fl_str_mv | Intracellular Ionic Strength Sensing Using NanoLuc |
| dc.type.none.fl_str_mv | Text Journal contribution info:eu-repo/semantics/publishedVersion text contribution to journal |
| description | <div><p>Intracellular ionic strength regulates myriad cellular processes that are fundamental to cellular survival and proliferation, including protein activity, aggregation, phase separation, and cell volume. It could be altered by changes in the activity of cellular signaling pathways, such as those that impact the activity of membrane-localized ion channels or by alterations in the microenvironmental osmolarity. Therefore, there is a demand for the development of sensitive tools for real-time monitoring of intracellular ionic strength. Here, we developed a bioluminescence-based intracellular ionic strength sensing strategy using the Nano Luciferase (NanoLuc) protein that has gained tremendous utility due to its high, long-lived bioluminescence output and thermal stability. Biochemical experiments using a recombinantly purified protein showed that NanoLuc bioluminescence is dependent on the ionic strength of the reaction buffer for a wide range of ionic strength conditions. Importantly, the decrease in the NanoLuc activity observed at higher ionic strengths could be reversed by decreasing the ionic strength of the reaction, thus making it suitable for sensing intracellular ionic strength alterations. Finally, we used an mNeonGreen–NanoLuc fusion protein to successfully monitor ionic strength alterations in a ratiometric manner through independent fluorescence and bioluminescence measurements in cell lysates and live cells. We envisage that the biosensing strategy developed here for detecting alterations in intracellular ionic strength will be applicable in a wide range of experiments, including high throughput cellular signaling, ion channel functional genomics, and drug discovery.</p><p> </p></div><h2>Other Information</h2> <p> Published in: International Journal of Molecular Sciences<br> License: <a href="https://creativecommons.org/licenses/by/4.0/" target="_blank">https://creativecommons.org/licenses/by/4.0/</a><br>See article on publisher's website: <a href="https://dx.doi.org/10.3390/ijms22020677" target="_blank">https://dx.doi.org/10.3390/ijms22020677</a></p> |
| eu_rights_str_mv | openAccess |
| id | Manara2_6165213776fec69e1cf332c878d820dd |
| identifier_str_mv | 10.3390/ijms22020677 |
| network_acronym_str | Manara2 |
| network_name_str | Manara2 |
| oai_identifier_str | oai:figshare.com:article/25867678 |
| publishDate | 2021 |
| repository.mail.fl_str_mv | |
| repository.name.fl_str_mv | |
| repository_id_str | |
| rights_invalid_str_mv | CC BY 4.0 |
| spelling | Intracellular Ionic Strength Sensing Using NanoLucTausif Altamash (3846304)Wesam Ahmed (4870942)Saad Rasool (9764093)Kabir H. Biswas (5705864)Biological sciencesBiochemistry and cell biologybioluminescencebiosensorionic strengthNanoLuc<div><p>Intracellular ionic strength regulates myriad cellular processes that are fundamental to cellular survival and proliferation, including protein activity, aggregation, phase separation, and cell volume. It could be altered by changes in the activity of cellular signaling pathways, such as those that impact the activity of membrane-localized ion channels or by alterations in the microenvironmental osmolarity. Therefore, there is a demand for the development of sensitive tools for real-time monitoring of intracellular ionic strength. Here, we developed a bioluminescence-based intracellular ionic strength sensing strategy using the Nano Luciferase (NanoLuc) protein that has gained tremendous utility due to its high, long-lived bioluminescence output and thermal stability. Biochemical experiments using a recombinantly purified protein showed that NanoLuc bioluminescence is dependent on the ionic strength of the reaction buffer for a wide range of ionic strength conditions. Importantly, the decrease in the NanoLuc activity observed at higher ionic strengths could be reversed by decreasing the ionic strength of the reaction, thus making it suitable for sensing intracellular ionic strength alterations. Finally, we used an mNeonGreen–NanoLuc fusion protein to successfully monitor ionic strength alterations in a ratiometric manner through independent fluorescence and bioluminescence measurements in cell lysates and live cells. We envisage that the biosensing strategy developed here for detecting alterations in intracellular ionic strength will be applicable in a wide range of experiments, including high throughput cellular signaling, ion channel functional genomics, and drug discovery.</p><p> </p></div><h2>Other Information</h2> <p> Published in: International Journal of Molecular Sciences<br> License: <a href="https://creativecommons.org/licenses/by/4.0/" target="_blank">https://creativecommons.org/licenses/by/4.0/</a><br>See article on publisher's website: <a href="https://dx.doi.org/10.3390/ijms22020677" target="_blank">https://dx.doi.org/10.3390/ijms22020677</a></p>2021-01-12T03:00:00ZTextJournal contributioninfo:eu-repo/semantics/publishedVersiontextcontribution to journal10.3390/ijms22020677https://figshare.com/articles/journal_contribution/Intracellular_Ionic_Strength_Sensing_Using_NanoLuc/25867678CC BY 4.0info:eu-repo/semantics/openAccessoai:figshare.com:article/258676782021-01-12T03:00:00Z |
| spellingShingle | Intracellular Ionic Strength Sensing Using NanoLuc Tausif Altamash (3846304) Biological sciences Biochemistry and cell biology bioluminescence biosensor ionic strength NanoLuc |
| status_str | publishedVersion |
| title | Intracellular Ionic Strength Sensing Using NanoLuc |
| title_full | Intracellular Ionic Strength Sensing Using NanoLuc |
| title_fullStr | Intracellular Ionic Strength Sensing Using NanoLuc |
| title_full_unstemmed | Intracellular Ionic Strength Sensing Using NanoLuc |
| title_short | Intracellular Ionic Strength Sensing Using NanoLuc |
| title_sort | Intracellular Ionic Strength Sensing Using NanoLuc |
| topic | Biological sciences Biochemistry and cell biology bioluminescence biosensor ionic strength NanoLuc |