Open AccessAn optogenetic method for interrogating YAP1 and TAZ nuclear–cytoplasmic shuttling
Author(s) -
Anna M. Dowbaj,
Robert P. Jenkins,
Daniel Williamson,
John M. Heddleston,
Alessandro Ciccarelli,
Todd Fallesen,
Klaus M. Hahn,
Reuben D. O’Dea,
John R. King,
Marco Montagner,
Erik Sahai
Publication year - 2021
Publication title -
journal of cell science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.384
H-Index - 278
eISSN - 1477-9137
pISSN - 0021-9533
DOI - 10.1242/jcs.253484
Subject(s) - yap1 , biology , nuclear export signal , cytoplasm , microbiology and biotechnology , nucleus , hippo signaling pathway , nuclear transport , sumo protein , optogenetics , degron , nuclear localization sequence , transcription factor , cell nucleus , biophysics , signal transduction , biochemistry , ubiquitin , gene , neuroscience , ubiquitin ligase
The shuttling of transcription factors and transcriptional regulators into and out of the nucleus is central to the regulation of many biological processes. Here we describe a new method for studying the rates of nuclear entry and exit of transcriptional regulators. A photo-responsive LOV (light-oxygen-voltage) domain from Avena sativa is used to sequester fluorescently labelled transcriptional regulators YAP1 and TAZ (also known as WWTR1) on the surface of mitochondria and to reversibly release them upon blue light illumination. After dissociation, fluorescent signals from the mitochondria, cytoplasm and nucleus are extracted by a bespoke app and used to generate rates of nuclear entry and exit. Using this method, we demonstrate that phosphorylation of YAP1 on canonical sites enhances its rate of nuclear export. Moreover, we provide evidence that, despite high intercellular variability, YAP1 import and export rates correlate within the same cell. By simultaneously releasing YAP1 and TAZ from sequestration, we show that their rates of entry and exit are correlated. Furthermore, combining the optogenetic release of YAP1 with lattice light-sheet microscopy reveals high heterogeneity of YAP1 dynamics within different cytoplasmic regions, demonstrating the utility and versatility of our tool to study protein dynamics. This article has an associated First Person interview with Anna M. Dowbaj, joint first author of the paper.