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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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)