A composition-dependent molecular clutch between T cell signaling condensates and actin
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
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.
Reviewing Editor
- Anthony A Hyman, Max Planck Institute of Molecular Cell Biology and Genetics, Germany
Publication history
- Received: October 9, 2018
- Accepted: June 14, 2019
- Accepted Manuscript published: July 3, 2019 (version 1)
- Version of Record published: July 11, 2019 (version 2)
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
-
- 4,383
- Page views
-
- 756
- Downloads
-
- 51
- Citations
Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.
Download links
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)
Further reading
-
- Biochemistry and Chemical Biology
- Cell Biology
Dystroglycan (DG) requires extensive post-translational processing and O-glycosylation to function as a receptor for extracellular matrix (ECM) proteins containing laminin-G-like (LG) domains. Matriglycan is an elongated polysaccharide of alternating xylose (Xyl) and glucuronic acid (GlcA) that binds with high-affinity to ECM proteins with LG-domains and is uniquely synthesized on α-dystroglycan (α-DG) by like-acetylglucosaminyltransferase-1 (LARGE1). Defects in the post-translational processing or O-glycosylation of α-DG that result in a shorter form of matriglycan reduce the size of α-DG and decrease laminin binding, leading to various forms of muscular dystrophy. Previously, we demonstrated that Protein O-Mannose Kinase (POMK) is required for LARGE1 to generate full-length matriglycan on α-DG (~150-250 kDa) (Walimbe et al., 2020). Here, we show that LARGE1 can only synthesize a short, non-elongated form of matriglycan in mouse skeletal muscle that lacks the DG N-terminus (α-DGN), resulting in a ~100-125 kDa α-DG. This smaller form of α-DG binds laminin and maintains specific force but does not prevent muscle pathophysiology, including reduced force production after eccentric contractions or abnormalities in the neuromuscular junctions. Collectively, our study demonstrates that α-DGN, like POMK, is required for LARGE1 to extend matriglycan to its full mature length on α-DG and thus prevent muscle pathophysiology.
-
- Biochemistry and Chemical Biology
- Microbiology and Infectious Disease
The malaria parasite Plasmodium falciparum synthesizes significant amounts of phospholipids to meet the demands of replication within red blood cells. De novo phosphatidylcholine (PC) biosynthesis via the Kennedy pathway is essential, requiring choline that is primarily sourced from host serum lysophosphatidylcholine (lysoPC). LysoPC also acts as an environmental sensor to regulate parasite sexual differentiation. Despite these critical roles for host lysoPC, the enzyme(s) involved in its breakdown to free choline for PC synthesis are unknown. Here, we show that a parasite glycerophosphodiesterase (PfGDPD) is indispensable for blood stage parasite proliferation. Exogenous choline rescues growth of PfGDPD-null parasites, directly linking PfGDPD function to choline incorporation. Genetic ablation of PfGDPD reduces choline uptake from lysoPC, resulting in depletion of several PC species in the parasite, whilst purified PfGDPD releases choline from glycerophosphocholine in vitro. Our results identify PfGDPD as a choline-releasing glycerophosphodiesterase that mediates a critical step in PC biosynthesis and parasite survival.