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25_Analysis_of_membrane_Piezo1_kinetics_using_GenEPi-A_new_tool_for_non-invasive_imaging_of_Piezo1_activity_CBIAS2022.pdf (3.13 MB)

Analysis of membrane Piezo1 kinetics using GenEPi - A new tool for non-invasive imaging of Piezo1 activity

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posted on 2023-03-03, 14:20 authored by Lorena Garcia-Foncillas, Fotini Kioutsouki, Konstantinos KalyviotisKonstantinos Kalyviotis, Periklis Pantazis

Poster presented as part of the Crick BioImage Analysis Symposium. 

Mechanosensation is a fundamental biological process in which mechanical stimuli are translated into complex biochem-ical and cellular responses. Piezo1, an essential mechanosensitive ion channel, has been identified to play a criticalrole in many physiological processes. Previously, fluorescent tagging of Piezo1 has been used to monitor the mem-brane localization of Piezo1 channels. However, the channel’s activity, i.e., when the channel is open and Ca2+ flowsthrough, cannot be reported by such approaches. We recently developed GenEPi, a genetically encoded fluorescentreporter of Piezo1 activity, which simultaneously reports the localization and activity of Piezo1 clusters captured usingTotal Internal Reflection Fluorescence (TIRF) microscopy. Here, we used Trackmate/Fiji and Python cell segmentationand tracking pipelines to track individual GenEPi clusters in the cell membrane of HEK293T cells. We used the producedtracks (TrackMate - 4110 tracks, Python - 940 tracks) to perform diffusion analyses using (i) Mean Squared Displace-ment (MSD) and (ii) Jump Distance Distribution (JDD). After testing different diffusion models, such as pure, directed,or anomalous diffusion, and their combinations, we found that Piezo1 presents an anomalous diffusion, with an averagediffusion coefficient of 0.0004μm2/s, in the range of previously reported values for wt-Piezo1. Future analyses of Piezo1cluster kinetics and connection to their activity profiles using GenEPi will enable us to reveal the complex role of Piezo1in cell physiology and homeostasis.

Permission has been given by authors to upload to Crick Figshare. Copyright remains with the original holders. 

Funding

Royal Society Wolfson Research Merit Award

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