Maib, H. orcid.org/0000-0002-1714-5746, Adarska, P. orcid.org/0000-0002-4407-4277, Hunton, R. orcid.org/0000-0001-7230-1871 et al. (4 more authors) (2024) Recombinant biosensors for multiplex and super-resolution imaging of phosphoinositides. Journal of Cell Biology, 223 (6). e202310095. ISSN 0021-9525
Abstract
Phosphoinositides are a small family of phospholipids that act as signaling hubs and key regulators of cellular function. Detecting their subcellular distribution is crucial to gain insights into membrane organization and is commonly done by the overexpression of biosensors. However, this leads to cellular perturbations and is challenging in systems that cannot be transfected. Here, we present a toolkit for the reliable, fast, multiplex, and super-resolution detection of phosphoinositides in fixed cells and tissue, based on recombinant biosensors with self-labeling SNAP tags. These are highly specific and reliably visualize the subcellular distributions of phosphoinositides across scales, from 2D or 3D cell culture to Drosophila tissue. Further, these probes enable super-resolution approaches, and using STED microscopy, we reveal the nanoscale organization of PI(3)P on endosomes and PI(4)P on the Golgi. Finally, multiplex staining reveals an unexpected presence of PI(3,5)P2-positive membranes in swollen lysosomes following PIKfyve inhibition. This approach enables the versatile, high-resolution visualization of multiple phosphoinositide species in an unprecedented manner.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2024 Maib et al. This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/). |
Keywords: | Endosomes; Phosphatidylinositol Phosphates; Phosphatidylinositols; Biosensing Techniques |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Science (Sheffield) > School of Biosciences (Sheffield) |
Funding Information: | Funder Grant number WELLCOME TRUST (THE) 210630/Z/18/Z Medical Research Council MR/X012077/1 |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 15 Apr 2024 14:37 |
Last Modified: | 15 Apr 2024 14:37 |
Status: | Published |
Publisher: | Rockefeller University Press |
Refereed: | Yes |
Identification Number: | 10.1083/jcb.202310095 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:211520 |
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