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Published September 22, 2023 | Version v1
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High-content imaging, immunoblot and immunofluorescence data related to: Caprin-1 binding to the critical stress granule protein G3BP1 is influenced by pH

Authors/Creators

  • 1. Karolinska Institute
  • 2. Harvard Medical School
  • 3. Royal Institute of Technology

Description

G3BP is the central node within stress-induced protein–RNA interaction networks known as stress granules (SGs). The SG-associated proteins Caprin-1 and USP10 bind mutually exclusively to the NTF2 domain of G3BP1, promoting and inhibiting SG formation, respectively. Herein, we present the crystal structure of G3BP1-NTF2 in complex with a Caprin-1-derived short linear motif (SLiM). Caprin-1 interacts with His-31 and His-62 within a third NTF2-binding site outside those covered by USP10, as confirmed using biochemical and biophysical-binding assays. Nano-differential scanning fluorimetry revealed reduced thermal stability of G3BP1-NTF2 at acidic pH. This destabilization was counterbalanced significantly better by bound USP10 than Caprin-1. The G3BP1/USP10 complex immunoprecipated from human U2OS cells was more resistant to acidic buffer washes than G3BP1/Caprin-1. Acidification of cellular condensates by approximately 0.5 units relative to the cytosol was detected by ratiometric fluorescence analysis of pHluorin2 fused to G3BP1. Cells expressing a Caprin-1/FGDF chimera with higher G3BP1-binding affinity had reduced Caprin-1 levels and slightly reduced condensate sizes. This unexpected finding may suggest that binding of the USP10-derived SLiM to NTF2 reduces the propensity of G3BP1 to enter condensates.

Notes

See README file for full details.

general notes on deposited file formats:
*.TIF or *.tif image files can be opened using standard software for images (ImageJ, Photoshop,...)
*.xlsx can be opened using Microsoft Excel
*.cpproj project files can be opened in Cellprofiler (https://cellprofiler.org/)

High-throughput (HT) imaging datasets can be analysed by opening the deposited Cellprofiler pipelines in Cellprofiler (https://cellprofiler.org/).
TIF files can also be viewed using any image viewing software (https://imagej.nih.gov/ij/download.html).

Scripts to analyse the Cellprofiler output, and additional information is found on Github:

https://github.com/derpaule/RSOB-22-0369

 

Funding provided by: Swedish Research Council
Crossref Funder Registry ID: http://dx.doi.org/10.13039/501100004359
Award Number: 2018-03843

Funding provided by: Swedish Research Council
Crossref Funder Registry ID: http://dx.doi.org/10.13039/501100004359
Award Number: 2018-03914

Funding provided by: Swedish Cancer Foundation
Crossref Funder Registry ID: http://dx.doi.org/10.13039/100012538
Award Number: CAN 2018/829

Funding provided by: Swedish Cancer Foundation
Crossref Funder Registry ID: http://dx.doi.org/10.13039/100012538
Award Number: CF 2018/603

Funding provided by: Swedish Research Council
Crossref Funder Registry ID: http://dx.doi.org/10.13039/501100004359
Award Number: 2018-02874

Funding provided by: Svenska Sällskapet för Medicinsk Forskning
Crossref Funder Registry ID: http://dx.doi.org/10.13039/501100003748
Award Number: P16-0083

Funding provided by: Stiftelsen Clas Groschinskys Minnesfond
Crossref Funder Registry ID: http://dx.doi.org/10.13039/501100006350
Award Number: M2002

Funding provided by: Swedish Cancer Foundation
Crossref Funder Registry ID: http://dx.doi.org/10.13039/100012538
Award Number: 2018/603

Funding provided by: Swedish Cancer Foundation
Crossref Funder Registry ID: http://dx.doi.org/10.13039/100012538
Award Number: 2018/829

Funding provided by: National Institutes of Health
Crossref Funder Registry ID: http://dx.doi.org/10.13039/100000002
Award Number: GM126901

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