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Observing Signaling Dynamics at Endogenous Levels Using a New Class of Biosensors FluoSTEP
Author(s) -
Zhang Jason Zhaoxing,
Tenner Brian,
Huang Bo,
Mehta Sohum,
Zhang Jin
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.686.3
Subject(s) - gtpase , förster resonance energy transfer , biosensor , microbiology and biotechnology , context (archaeology) , signal transduction , biology , small gtpase , chemistry , biochemistry , fluorescence , paleontology , physics , quantum mechanics
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. Support or Funding Information NIH R01 DK073368 and R35 CA197622 (to JZ), NSF GRFP (to JZZ) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .