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Single-molecule localization microscopy reveals STING clustering at the trans-Golgi network through palmitoylation-dependent accumulation of cholesterol.

ABSTRACT: Stimulator of interferon genes (STING) is critical for the type I interferon response to pathogen- or self-derived DNA in the cytosol. STING may function as a scaffold to activate TANK-binding kinase 1 (TBK1), but direct cellular evidence remains lacking. Here we show, using single-molecule imaging of STING with enhanced time resolutions down to 5 ms, that STING becomes clustered at the trans-Golgi network (about 20 STING molecules per cluster). The clustering requires STING palmitoylation and the Golgi lipid order defined by cholesterol. Single-molecule imaging of TBK1 reveals that STING clustering enhances the association with TBK1. We thus provide quantitative proof-of-principle for the signaling STING scaffold, reveal the mechanistic role of STING palmitoylation in the STING activation, and resolve the long-standing question of the requirement of STING translocation for triggering the innate immune signaling.

SUBMITTER: Kemmoku H

PROVIDER: S-EPMC10784591 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

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Single-molecule localization microscopy reveals STING clustering at the trans-Golgi network through palmitoylation-dependent accumulation of cholesterol.

Kemmoku Haruka H Takahashi Kanoko K Mukai Kojiro K Mori Toshiki T Hirosawa Koichiro M KM Kiku Fumika F Uchida Yasunori Y Kuchitsu Yoshihiko Y Nishioka Yu Y Sawa Masaaki M Kishimoto Takuma T Tanaka Kazuma K Yokota Yasunari Y Arai Hiroyuki H Suzuki Kenichi G N KGN Taguchi Tomohiko T

Nature communications 20240111 1

Stimulator of interferon genes (STING) is critical for the type I interferon response to pathogen- or self-derived DNA in the cytosol. STING may function as a scaffold to activate TANK-binding kinase 1 (TBK1), but direct cellular evidence remains lacking. Here we show, using single-molecule imaging of STING with enhanced time resolutions down to 5 ms, that STING becomes clustered at the trans-Golgi network (about 20 STING molecules per cluster). The clustering requires STING palmitoylation and t ...[more]

PMID: 38212328

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Project description:Ligand binding of membrane proteins triggers many important cellular signaling events by the lateral aggregation of ligand-bound and other membrane proteins in the plane of the plasma membrane. This local clustering can lead to the co-enrichment of molecules that create an intracellular signal or bring sufficient amounts of activity together to shift an existing equilibrium towards the execution of a signaling event. In this way, clustering can serve as a cellular switch. The underlying uneven distribution and local enrichment of the signaling cluster's constituting membrane proteins can be used as a functional readout. This information is obtained by combining single-molecule fluorescence microscopy with cluster algorithms that can reliably and reproducibly distinguish clusters from fluctuations in the background noise to generate quantitative data on this complex process. Cluster analysis of single-molecule fluorescence microscopy data has emerged as a proliferative field, and several algorithms and software solutions have been put forward. However, in most cases, such cluster algorithms require multiple analysis parameters to be defined by the user, which may lead to biased results. Furthermore, most cluster algorithms neglect the individual localization precision connected to every localized molecule, leading to imprecise results. Bayesian cluster analysis has been put forward to overcome these problems, but so far, it has entailed high computational cost, increasing runtime drastically. Finally, most software is challenging to use as they require advanced technical knowledge to operate. Here we combined three advanced cluster algorithms with the Bayesian approach and parallelization in a user-friendly GUI and achieved up to an order of magnitude faster processing than for previous approaches. Our work will simplify access to a well-controlled analysis of clustering data generated by SMLM and significantly accelerate data processing. The inclusion of a simulation mode aids in the design of well-controlled experimental assays.

Dataset Information

Single-molecule localization microscopy reveals STING clustering at the trans-Golgi network through palmitoylation-dependent accumulation of cholesterol.

Publications

Single-molecule localization microscopy reveals STING clustering at the trans-Golgi network through palmitoylation-dependent accumulation of cholesterol.

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