A Novel NIR-FRET Biosensor for Reporting PS/?-Secretase Activity in Live Cells.

ABSTRACT: Presenilin (PS)/?-secretase plays a pivotal role in essential cellular events via proteolytic processing of transmembrane proteins that include APP and Notch receptors. However, how PS/?-secretase activity is spatiotemporally regulated by other molecular and cellular factors and how the changes in PS/?-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/?-secretase activity and additional cellular events in real-time. Here, we report the development of a near-infrared (NIR) FRET-based PS/?-secretase biosensor, C99 720-670 probe, which incorporates an immediate PS/?-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/?-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), ** p < 0.01). Importantly, the C99 720-670 and the previously developed APP C99 YPet-Turquoise-GL (C99 Y-T) biosensors simultaneously report PS/?-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/?-secretase activity.

SUBMITTER: Houser MC 

PROVIDER: S-EPMC7660074 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature