1. Biochemistry and Chemical Biology
  2. Cell Biology

A composition-dependent molecular clutch between T cell signaling condensates and actin

  1. Jonathon A Ditlev
  2. Anthony R Vega
  3. Darius Vasco Köster
  4. Xiaolei Su
  5. Tomomi Tani
  6. Ashley M Lakoduk
  7. Ronald D Vale
  8. Satyajit Mayor  Is a corresponding author
  9. Khuloud Jaqaman  Is a corresponding author
  10. Michael K Rosen  Is a corresponding author
  1. Marine Biological Laboratory, United States
  2. University of Texas Southwestern Medical Center, United States
https://doi.org/10.7554/eLife.42695
Share this article
Cite this article
  1. Jonathon A Ditlev
  2. Anthony R Vega
  3. Darius Vasco Köster
  4. Xiaolei Su
  5. Tomomi Tani
  6. Ashley M Lakoduk
  7. Ronald D Vale
  8. Satyajit Mayor
  9. Khuloud Jaqaman
  10. Michael K Rosen
(2019)
A composition-dependent molecular clutch between T cell signaling condensates and actin
eLife 8:e42695.
https://doi.org/10.7554/eLife.42695
  2. Download BibTeX
  3. Download .RIS

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.

The following data sets were generated

Article and author information

Author details

  1. Jonathon A Ditlev

    Howard Hughes Medical Institute Summer Institute, Marine Biological Laboratory, Woods Hole, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8287-7700

  2. Anthony R Vega

    Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4464-6482

  3. Darius Vasco Köster

    Howard Hughes Medical Institute Summer Institute, Marine Biological Laboratory, Woods Hole, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8530-5476

  4. Xiaolei Su

    Howard Hughes Medical Institute Summer Institute, Marine Biological Laboratory, Woods Hole, United States
    Competing interests
    The authors declare that no competing interests exist.

  5. Tomomi Tani

    Eugene Bell Center for Regenerative Biology and Tissue Engineering, Marine Biological Laboratory, Woods Hole, United States
    Competing interests
    The authors declare that no competing interests exist.

  6. Ashley M Lakoduk

    Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    The authors declare that no competing interests exist.

  7. Ronald D Vale

    Howard Hughes Medical Institute Summer Institute, Marine Biological Laboratory, Woods Hole, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3460-2758

  8. Satyajit Mayor

    Howard Hughes Medical Institute Summer Institute, Marine Biological Laboratory, Woods Hole, United States
    For correspondence
    mayor@ncbs.res.in
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9842-6963

  9. Khuloud Jaqaman

    Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States
    For correspondence
    khuloud.jaqaman@utsouthwestern.edu
    Competing interests
    The authors declare that no competing interests exist.

  10. Michael K Rosen

    Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States
    For correspondence
    michael.rosen@utsouthwestern.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0775-7917

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

  1. Anthony A Hyman, Max Planck Institute of Molecular Cell Biology and Genetics, Germany

Publication history

  1. Received: October 9, 2018
  2. Accepted: June 14, 2019
  3. Accepted Manuscript published: July 3, 2019 (version 1)
  4. 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

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)

  1. Jonathon A Ditlev
  2. Anthony R Vega
  3. Darius Vasco Köster
  4. Xiaolei Su
  5. Tomomi Tani
  6. Ashley M Lakoduk
  7. Ronald D Vale
  8. Satyajit Mayor
  9. Khuloud Jaqaman
  10. Michael K Rosen
(2019)
A composition-dependent molecular clutch between T cell signaling condensates and actin
eLife 8:e42695.
https://doi.org/10.7554/eLife.42695

Categories and tags

Research organism

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics

    RNA sequence to structure analysis from comprehensive pairwise mutagenesis of multiple self-cleaving ribozymes

    Jessica M Roberts, James D Beck ... Eric J Hayden
    Research Article Updated

    Self-cleaving ribozymes are RNA molecules that catalyze the cleavage of their own phosphodiester backbones. These ribozymes are found in all domains of life and are also a tool for biotechnical and synthetic biology applications. Self-cleaving ribozymes are also an important model of sequence-to-function relationships for RNA because their small size simplifies synthesis of genetic variants and self-cleaving activity is an accessible readout of the functional consequence of the mutation. Here, we used a high-throughput experimental approach to determine the relative activity for every possible single and double mutant of five self-cleaving ribozymes. From this data, we comprehensively identified non-additive effects between pairs of mutations (epistasis) for all five ribozymes. We analyzed how changes in activity and trends in epistasis map to the ribozyme structures. The variety of structures studied provided opportunities to observe several examples of common structural elements, and the data was collected under identical experimental conditions to enable direct comparison. Heatmap-based visualization of the data revealed patterns indicating structural features of the ribozymes including paired regions, unpaired loops, non-canonical structures, and tertiary structural contacts. The data also revealed signatures of functionally critical nucleotides involved in catalysis. The results demonstrate that the data sets provide structural information similar to chemical or enzymatic probing experiments, but with additional quantitative functional information. The large-scale data sets can be used for models predicting structure and function and for efforts to engineer self-cleaving ribozymes.

    1. Biochemistry and Chemical Biology
    2. Cell Biology

    N-terminal domain on dystroglycan enables LARGE1 to extend matriglycan on α-dystroglycan and prevents muscular dystrophy

    Hidehiko Okuma, Jeffrey M Hord ... Kevin P Campbell
    Research Advance

    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.

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease

    A choline-releasing glycerophosphodiesterase essential for phosphatidylcholine biosynthesis and blood stage development in the malaria parasite

    Abhinay Ramaprasad, Paul-Christian Burda ... Michael J Blackman
    Research Article Updated

    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.