An optogenetic method for interrogating YAP1 and TAZ nuclear-cytoplasmic shuttling
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Anna Dowbaj Rob Jenkins Daniel Williamson John M Heddleston Alessandro Ciccarelli Todd Fallesen Klaus M Hahn Reuben D O'Dea John R King Marco Montagner Erik SahaiAbstract
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 AsLOV (Avena sativa Light Oxygen Voltage) domain is used to sequester fluorescently-labelled transcriptional regulators YAP1 and TAZ/WWTR1 on the surface of mitochondria and reversibly release them upon blue light illumination. After dissociation, fluorescent signals from mitochondria, cytoplasm and nucleus are extracted with 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 correlated 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 revealed high heterogeneity of YAP1 dynamics within different cytoplasmic regions, demonstrating the utility and versatility of our tool to study protein dynamics.
Journal details
Journal
Journal of Cell Science
Volume
134
Issue number
13
Pages
jcs253484
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10.1242/jcs.253484
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Europe PubMed Central
34060624
Pubmed
34060624
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