A Novel NIR-FRET Biosensor for Reporting PS/γ-Secretase Activity in Live Cells
Presenilin (PS)/γ-secretase plays a pivotal role in essential cellular events via proteolytic processing of transmembrane proteins that include APP and Notch receptors. However, how PS/γ-secretase activity is spatiotemporally regulated by other molecular and cellular factors and how the changes in P...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2020-10-01
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| Series: | Sensors |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1424-8220/20/21/5980 |
| _version_ | 1797550158307655680 |
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| author | Mei CQ Houser Steven S Hou Florian Perrin Yuliia Turchyna Brian J Bacskai Oksana Berezovska Masato Maesako |
| author_facet | Mei CQ Houser Steven S Hou Florian Perrin Yuliia Turchyna Brian J Bacskai Oksana Berezovska Masato Maesako |
| author_sort | Mei CQ Houser |
| collection | DOAJ |
| description | Presenilin (PS)/γ-secretase plays a pivotal role in essential cellular events via proteolytic processing of transmembrane proteins that include APP and Notch receptors. However, how PS/γ-secretase activity is spatiotemporally regulated by other molecular and cellular factors and how the changes in PS/γ-secretase activity influence signaling pathways in live cells are poorly understood. These questions could be addressed by engineering a new tool that enables multiplexed imaging of PS/γ-secretase activity and additional cellular events in real-time. Here, we report the development of a near-infrared (NIR) FRET-based PS/γ-secretase biosensor, C99 720-670 probe, which incorporates an immediate PS/γ-secretase substrate APP C99 with miRFP670 and miRFP720 as the donor and acceptor fluorescent proteins, respectively. Extensive validation demonstrates that the C99 720-670 biosensor enables quantitative monitoring of endogenous PS/γ-secretase activity on a cell-by-cell basis in live cells (720/670 ratio: 2.47 ± 0.66 (vehicle) vs. 3.02 ± 1.17 (DAPT), ** <i>p</i> < 0.01). Importantly, the C99 720-670 and the previously developed APP C99 YPet-Turquoise-GL (C99 Y-T) biosensors simultaneously report PS/γ-secretase activity. This evidences the compatibility of the C99 720-670 biosensor with cyan (CFP)-yellow fluorescent protein (YFP)-based FRET biosensors for reporting other essential cellular events. Multiplexed imaging using the novel NIR biosensor C99 720-670 would open a new avenue to better understand the regulation and consequences of changes in PS/γ-secretase activity. |
| first_indexed | 2024-03-10T15:24:31Z |
| format | Article |
| id | doaj.art-53b08fde53de4967a8b63021b3383a3e |
| institution | Directory Open Access Journal |
| issn | 1424-8220 |
| language | English |
| last_indexed | 2024-03-10T15:24:31Z |
| publishDate | 2020-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Sensors |
| spelling | doaj.art-53b08fde53de4967a8b63021b3383a3e2023-11-20T18:07:46ZengMDPI AGSensors1424-82202020-10-012021598010.3390/s20215980A Novel NIR-FRET Biosensor for Reporting PS/γ-Secretase Activity in Live CellsMei CQ Houser0Steven S Hou1Florian Perrin2Yuliia Turchyna3Brian J Bacskai4Oksana Berezovska5Masato Maesako6Alzheimer Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, 114, 16th street, Charlestown, MA 02129, USAAlzheimer Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, 114, 16th street, Charlestown, MA 02129, USAAlzheimer Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, 114, 16th street, Charlestown, MA 02129, USAAlzheimer Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, 114, 16th street, Charlestown, MA 02129, USAAlzheimer Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, 114, 16th street, Charlestown, MA 02129, USAAlzheimer Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, 114, 16th street, Charlestown, MA 02129, USAAlzheimer Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, 114, 16th street, Charlestown, MA 02129, USAPresenilin (PS)/γ-secretase plays a pivotal role in essential cellular events via proteolytic processing of transmembrane proteins that include APP and Notch receptors. However, how PS/γ-secretase activity is spatiotemporally regulated by other molecular and cellular factors and how the changes in PS/γ-secretase activity influence signaling pathways in live cells are poorly understood. These questions could be addressed by engineering a new tool that enables multiplexed imaging of PS/γ-secretase activity and additional cellular events in real-time. Here, we report the development of a near-infrared (NIR) FRET-based PS/γ-secretase biosensor, C99 720-670 probe, which incorporates an immediate PS/γ-secretase substrate APP C99 with miRFP670 and miRFP720 as the donor and acceptor fluorescent proteins, respectively. Extensive validation demonstrates that the C99 720-670 biosensor enables quantitative monitoring of endogenous PS/γ-secretase activity on a cell-by-cell basis in live cells (720/670 ratio: 2.47 ± 0.66 (vehicle) vs. 3.02 ± 1.17 (DAPT), ** <i>p</i> < 0.01). Importantly, the C99 720-670 and the previously developed APP C99 YPet-Turquoise-GL (C99 Y-T) biosensors simultaneously report PS/γ-secretase activity. This evidences the compatibility of the C99 720-670 biosensor with cyan (CFP)-yellow fluorescent protein (YFP)-based FRET biosensors for reporting other essential cellular events. Multiplexed imaging using the novel NIR biosensor C99 720-670 would open a new avenue to better understand the regulation and consequences of changes in PS/γ-secretase activity.https://www.mdpi.com/1424-8220/20/21/5980Presenilin (PS)/γ-secretaseFRET biosensornear-infrared (NIR)miRFP670 and 720 |
| spellingShingle | Mei CQ Houser Steven S Hou Florian Perrin Yuliia Turchyna Brian J Bacskai Oksana Berezovska Masato Maesako A Novel NIR-FRET Biosensor for Reporting PS/γ-Secretase Activity in Live Cells Sensors Presenilin (PS)/γ-secretase FRET biosensor near-infrared (NIR) miRFP670 and 720 |
| title | A Novel NIR-FRET Biosensor for Reporting PS/γ-Secretase Activity in Live Cells |
| title_full | A Novel NIR-FRET Biosensor for Reporting PS/γ-Secretase Activity in Live Cells |
| title_fullStr | A Novel NIR-FRET Biosensor for Reporting PS/γ-Secretase Activity in Live Cells |
| title_full_unstemmed | A Novel NIR-FRET Biosensor for Reporting PS/γ-Secretase Activity in Live Cells |
| title_short | A Novel NIR-FRET Biosensor for Reporting PS/γ-Secretase Activity in Live Cells |
| title_sort | novel nir fret biosensor for reporting ps γ secretase activity in live cells |
| topic | Presenilin (PS)/γ-secretase FRET biosensor near-infrared (NIR) miRFP670 and 720 |
| url | https://www.mdpi.com/1424-8220/20/21/5980 |
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