A composition-dependent molecular clutch between T cell signaling condensates and actin
- Marine Biological Laboratory, United States
- University of Texas Southwestern Medical Center, United States
Abstract
During T cell activation, biomolecular condensates form at the immunological synapse (IS) through multivalency-driven phase separation of LAT, Grb2, Sos1, SLP-76, Nck, and WASP. These condensates move radially at the IS, traversing successive radially-oriented and concentric actin networks. To understand this movement, we biochemically reconstituted LAT condensates with actomyosin filaments. We found that basic regions of Nck and N-WASP/WASP promote association and co-movement of LAT condensates with actin, indicating conversion of weak individual affinities to high collective affinity upon phase separation. Condensates lacking these components were propelled differently, without strong actin adhesion. In cells, LAT condensates lost Nck as radial actin transitioned to the concentric network, and engineered condensates constitutively binding actin moved aberrantly. Our data show that Nck and WASP form a clutch between LAT condensates and actin in vitro and suggest that compositional changes may enable condensate movement by distinct actin networks in different regions of the IS.
Data availability
Data are available in the BioStudies database (http://www.ebi.ac.uk/biostudies) under accession number S-BIAD6. Image data are available in the Image Data Resource (IDR) (https://idr.openmicroscopy.org) under accession number idr0055. Condensate analysis code is available on GitHub at https://github.com/kjaqaman/CondensateAnalysis. Colocalization analysis code is available on GitHub at https://github.com/kjaqaman/ColocPt2Cont. Cluster tracking analysis code is available on GitHub at https://github.com/DanuserLab/u-track. Polarization microscopy analysis code is available on GitHub at https://github.com/mattersoflight/Instantaneous-PolScope.
Article and author information
Author details
Darius Vasco Köster
Funding
Howard Hughes Medical Institute
- Ronald D Vale
- Michael K Rosen
Cancer Research Institute
- Xiaolei Su
National Institutes of Health (R35 GM119619)
- Khuloud Jaqaman
National Institutes of Health (F32 DK101188)
- Jonathon A Ditlev
Welch Foundation (I-1544)
- Michael K Rosen
Department of Science and Technology, Government of India (J C Bose Fellowship)
- Satyajit Mayor
Margadarshi Fellowship from the Wellcome Trust - Department of Biotechnology, India Alliance (IA/M/15/1/502018)
- Satyajit Mayor
National Institutes of Health (R01 GM100160)
- Tomomi Tani
UT Southwestern Endowed Scholars Program
- Khuloud Jaqaman
National Research Service Award F32 (F32 DK101188)
- Jonathon A Ditlev
CPRIT Training Grant (RP140110 PI: Michael White)
- Anthony R Vega
AXA Research Fund and the National Centre for Biological Sciences, Tata Institute for Fundamental Research
- Darius Vasco Köster
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2019, Ditlev et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 5,051
- views
-
- 842
- downloads
-
- 90
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
A composition-dependent molecular clutch between T cell signaling condensates and actin
eLife 8:e42695.
https://doi.org/10.7554/eLife.42695
Further reading
-
- Biochemistry and Chemical Biology
- Stem Cells and Regenerative Medicine
Human induced pluripotent stem cells (hiPSCs) have great potential to be used as alternatives to embryonic stem cells (hESCs) in regenerative medicine and disease modelling. In this study, we characterise the proteomes of multiple hiPSC and hESC lines derived from independent donors and find that while they express a near-identical set of proteins, they show consistent quantitative differences in the abundance of a subset of proteins. hiPSCs have increased total protein content, while maintaining a comparable cell cycle profile to hESCs, with increased abundance of cytoplasmic and mitochondrial proteins required to sustain high growth rates, including nutrient transporters and metabolic proteins. Prominent changes detected in proteins involved in mitochondrial metabolism correlated with enhanced mitochondrial potential, shown using high-resolution respirometry. hiPSCs also produced higher levels of secreted proteins, including growth factors and proteins involved in the inhibition of the immune system. The data indicate that reprogramming of fibroblasts to hiPSCs produces important differences in cytoplasmic and mitochondrial proteins compared to hESCs, with consequences affecting growth and metabolism. This study improves our understanding of the molecular differences between hiPSCs and hESCs, with implications for potential risks and benefits for their use in future disease modelling and therapeutic applications.
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
Dynamic conformational and structural changes in proteins and protein complexes play a central and ubiquitous role in the regulation of protein function, yet it is very challenging to study these changes, especially for large protein complexes, under physiological conditions. Here, we introduce a novel isobaric crosslinker, Qlinker, for studying conformational and structural changes in proteins and protein complexes using quantitative crosslinking mass spectrometry. Qlinkers are small and simple, amine-reactive molecules with an optimal extended distance of ~10 Å, which use MS2 reporter ions for relative quantification of Qlinker-modified peptides derived from different samples. We synthesized the 2-plex Q2linker and showed that the Q2linker can provide quantitative crosslinking data that pinpoints key conformational and structural changes in biosensors, binary and ternary complexes composed of the general transcription factors TBP, TFIIA, and TFIIB, and RNA polymerase II complexes.
