Abstract
ABSTRACTThe 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.
Highlights
Dysphagia is associated with poorer outcomes [1, 2]
126 participants received Pharyngeal electrical stimulation (PES) or sham and had both a videofluoroscopy swallow studies (VFSS) completed at baseline and 2 weeks
Our findings with regards to PA scores agree with the overall conclusion from the original Swallowing Treatment using Electrical Pharyngeal Stimulation Trial (STEPS) study which did not show a significant change between groups for safety
Summary
Dysphagia is associated with poorer outcomes [1, 2]. Despite this, there are few proven treatments for post-stroke (PSD) dysphagia and a pressing need for more high-quality trials in order to assess which specific interventions are effective [3]. Pharyngeal electrical stimulation (PES) is a potential treatment for PSD and has been used in several published studies to date [7, 11,12,13]. The largest of these was the Swallowing Treatment using Electrical Pharyngeal Stimulation Trial (STEPS) [7]. We investigated the effect of PES on multiple measures of both speed and duration (timing) and efficiency (clearance) as well as safety (PAS) by conducting a retrospective analysis on the STEPS data
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