{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,18]],"date-time":"2026-01-18T11:28:49Z","timestamp":1768735729270,"version":"3.49.0"},"reference-count":60,"publisher":"MDPI AG","issue":"21","license":[{"start":{"date-parts":[[2020,10,22]],"date-time":"2020-10-22T00:00:00Z","timestamp":1603324800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["AG 44486"],"award-info":[{"award-number":["AG 44486"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["AG 15379"],"award-info":[{"award-number":["AG 15379"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100006312","name":"BrightFocus Foundation","doi-asserted-by":"publisher","award":["A2019056F"],"award-info":[{"award-number":["A2019056F"]}],"id":[{"id":"10.13039\/100006312","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Sensors"],"abstract":"<jats:p>Presenilin (PS)\/\u03b3-secretase plays a pivotal role in essential cellular events via proteolytic processing of transmembrane proteins that include APP and Notch receptors. However, how PS\/\u03b3-secretase activity is spatiotemporally regulated by other molecular and cellular factors and how the changes in PS\/\u03b3-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\/\u03b3-secretase activity and additional cellular events in real-time. Here, we report the development of a near-infrared (NIR) FRET-based PS\/\u03b3-secretase biosensor, C99 720-670 probe, which incorporates an immediate PS\/\u03b3-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\/\u03b3-secretase activity on a cell-by-cell basis in live cells (720\/670 ratio: 2.47 \u00b1 0.66 (vehicle) vs. 3.02 \u00b1 1.17 (DAPT), ** p &lt; 0.01). Importantly, the C99 720-670 and the previously developed APP C99 YPet-Turquoise-GL (C99 Y-T) biosensors simultaneously report PS\/\u03b3-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\/\u03b3-secretase activity.<\/jats:p>","DOI":"10.3390\/s20215980","type":"journal-article","created":{"date-parts":[[2020,10,22]],"date-time":"2020-10-22T20:51:00Z","timestamp":1603399860000},"page":"5980","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":19,"title":["A Novel NIR-FRET Biosensor for Reporting PS\/\u03b3-Secretase Activity in Live Cells"],"prefix":"10.3390","volume":"20","author":[{"given":"Mei CQ","family":"Houser","sequence":"first","affiliation":[{"name":"Alzheimer Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, 114, 16th street, Charlestown, MA 02129, USA"}]},{"given":"Steven S","family":"Hou","sequence":"additional","affiliation":[{"name":"Alzheimer Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, 114, 16th street, Charlestown, MA 02129, USA"}]},{"given":"Florian","family":"Perrin","sequence":"additional","affiliation":[{"name":"Alzheimer Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, 114, 16th street, Charlestown, MA 02129, USA"}]},{"given":"Yuliia","family":"Turchyna","sequence":"additional","affiliation":[{"name":"Alzheimer Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, 114, 16th street, Charlestown, MA 02129, USA"}]},{"given":"Brian J","family":"Bacskai","sequence":"additional","affiliation":[{"name":"Alzheimer Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, 114, 16th street, Charlestown, MA 02129, USA"}]},{"given":"Oksana","family":"Berezovska","sequence":"additional","affiliation":[{"name":"Alzheimer Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, 114, 16th street, Charlestown, MA 02129, USA"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1970-2462","authenticated-orcid":false,"given":"Masato","family":"Maesako","sequence":"additional","affiliation":[{"name":"Alzheimer Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, 114, 16th street, Charlestown, MA 02129, USA"}]}],"member":"1968","published-online":{"date-parts":[[2020,10,22]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"9","DOI":"10.2174\/156802608783334060","article-title":"Activity of gamma-secretase on substrates other than APP","volume":"8","year":"2008","journal-title":"Curr. 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