70. observing signaling dynamics at endogenous levels using a new class of biosensors fluostep

Department: Bioengineering
Faculty Advisor(s): Jin Zhang

Primary Student
Name: Jason Zhaoxing Zhang
Email: jzz002@ucsd.edu
Phone: 757-816-6883
Grad Year: 2020

Intracellular signaling is highly spatiotemporally regulated with specific kinetically-distinct microdomains. Fluorescent biosensors such as FRET-based kinase activity reporter, second messenger indicators, and GTPase activation biosensors have facilitated the elucidation of the spatiotemporal regulation for various signaling protein networks; however, overexpression of these biosensors could perturb endogenous signaling. This is especially problematic for GTPase activation biosensors as they contain the GTPase within the sensing domain, thus, causing artificially high GTPase signaling activity. To overcome these challenges, we designed a novel class of fluorescent biosensors named Fluorescent Sensors Targeted to Endogenous Proteins (FluoSTEPs) that utilizes a split fluorescent protein approach to target a specific protein and reconstitute a functional sensor. We have shown that this design is generalizable for various FRET?based biosensors. Furthermore, combining this design with CRISPR knock-in technology, we demonstrated the application of FluoSTEP in studying cAMP and PKA signaling in the native clathrin-coated pits and Sec61B microdomains. With the capability of reporting endogenous signaling in the native context of a living cell, FluoSTEP will be powerful tools to investigate spatiotemporal regulation of signaling enzymes such as kinases and GTPases.

Industry Application Area(s)
Life Sciences/Medical Devices & Instruments

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