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Optogenetic activation and inactivation of the neurotrophin pathway in live cells
Author(s) -
Mondal Payel,
Krishnamurthy Vishnu Vardhan,
Sharum Savanna,
Haack Neeka,
Zhang Kai
Publication year - 2019
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.2019.33.1_supplement.476.18
Subject(s) - optogenetics , mapk/erk pathway , degron , signal transduction , microbiology and biotechnology , genetic screen , biology , hek 293 cells , crosstalk , phenotype , chemistry , neuroscience , cell culture , gene , genetics , ubiquitin ligase , physics , ubiquitin , optics
Signaling pathways extensively crosstalk among each other and result in different cellular phenotypes depending on the dynamic profile of protein activity. Conventional genetic and pharmacological approaches such as gene overexpression, use growth factors or inhibitors have helped us understand different aspects of signaling pathways, however these techniques provide limited means to determine contribution of a target protein for specific cellular phenotype. Therefore, to find out the molecular mechanism for a cellular outcome, there is an urgent need for a tool that can specifically activate or inactivate a protein of interest and study it's role towards a specific cell fate. Optogenetic techniques utilize light to control protein functions with high spatial and temporal resolution. Here we introduce a generalizable method for optical control of protein stability that enables fine spatiotemporal control of specific signaling pathways. This optogenetic system has been used to re‐purpose the protein degradation machinery by combining a small peptide sequence called degron, an evolved LOV (eLOV) protein and a light‐inducible nuclear export system (LEXY). Our system has been used to activate the Raf/MEK/ERK signaling cascade by light induced stabilization of a constitutively active cytosolic MEK (CA MEK). Light induced activation of the ERK pathway results in elevated phosphor‐ERK levels in human embryonic kidney cells (HEK) and rat PC12 pheochromocytoma cells as well as PC12 neurite outgrowth in the absence of growth factors. Furthermore, current non‐neuronal optogenetic methods provide fewer tools for inactivation of a specific signaling pathway compared to optogenetic activation. To bridge this gap, we have demonstrated inhibition of the ERK pathway by light induced stabilization of mitogen activated protein kinase phosphatase 3 (MKP3), an inhibitor of the ERK pathway. Thus, our system allows light‐mediated rescue of protein stability by using a degradation‐rescue strategy and offers the potential for regulation of a plethora of biological processes. Support or Funding Information This work was supported by the University of Illinois at Urbana‐Champaign (UIUC) (K.Z.) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .