1. Cell Biology
  2. Structural Biology and Molecular Biophysics

Detecting molecular interactions in live-cell single-molecule imaging with proximity-assisted photoactivation (PAPA)

  1. Thomas George Wade Graham
  2. John Joseph Ferrie III
  3. Gina M Dailey
  4. Robert Tjian  Is a corresponding author
  5. Xavier Darzacq  Is a corresponding author
  1. University of California, Berkeley, United States
  2. Howard Hughes Medical Institute, University of California, Berkeley, United States
https://doi.org/10.7554/eLife.76870
Share this article
Cite this article
  1. Thomas George Wade Graham
  2. John Joseph Ferrie III
  3. Gina M Dailey
  4. Robert Tjian
  5. Xavier Darzacq
(2022)
Detecting molecular interactions in live-cell single-molecule imaging with proximity-assisted photoactivation (PAPA)
eLife 11:e76870.
https://doi.org/10.7554/eLife.76870
  2. Download BibTeX
  3. Download .RIS

Abstract

Single-molecule imaging provides a powerful way to study biochemical processes in live cells, yet it remains challenging to track single molecules while simultaneously detecting their interactions. Here we describe a novel property of rhodamine dyes, proximity-assisted photoactivation (PAPA), in which one fluorophore (the 'sender') can reactivate a second fluorophore (the 'receiver') from a dark state. PAPA requires proximity between the two fluorophores, yet it operates at a longer average intermolecular distance than Förster resonance energy transfer (FRET). We show that PAPA can be used in live cells both to detect protein-protein interactions and to highlight a sub-population of labeled protein complexes in which two different labels are in proximity. In proof-of-concept experiments, PAPA detected the expected correlation between androgen receptor self-association and chromatin binding at the single-cell level. These results establish a new way in which a photophysical property of fluorophores can be harnessed to study molecular interactions in single-molecule imaging of live cells.

Data availability

Source data for Fig. 2-5 are included in an accompanying zip file.

Article and author information

Author details

  1. Thomas George Wade Graham

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    Thomas George Wade Graham, is an inventor on a pending patent application (PCT/US2021/062616) related to the use of PAPA as a molecular proximity sensor..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5189-4313

  2. John Joseph Ferrie III

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.

  3. Gina M Dailey

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8988-963X

  4. Robert Tjian

    Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States
    For correspondence
    tijcal@berkeley.edu
    Competing interests
    Robert Tjian, is a member of eLife's Board of Directors.Is a co-founder of Eikon Therapeutics, Inc.Is an inventor on a pending patent application (PCT/US2021/062616) related to the use of PAPA as a molecular proximity sensor..

  5. Xavier Darzacq

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    For correspondence
    darzacq@berkeley.edu
    Competing interests
    Xavier Darzacq, is a co-founder of Eikon Therapeutics, Inc.Is an inventor on a pending patent application (PCT/US2021/062616) related to the use of PAPA as a molecular proximity sensor..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2537-8395

Funding

Howard Hughes Medical Institute

  • Robert Tjian

Jane Coffin Childs Memorial Fund for Medical Research

  • Thomas George Wade Graham

Life Sciences Research Foundation

  • John Joseph Ferrie III

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2022, Graham 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

  • 8,398
    views
  • 1,343
    downloads
  • 27
    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)

  1. Thomas George Wade Graham
  2. John Joseph Ferrie III
  3. Gina M Dailey
  4. Robert Tjian
  5. Xavier Darzacq
(2022)
Detecting molecular interactions in live-cell single-molecule imaging with proximity-assisted photoactivation (PAPA)
eLife 11:e76870.
https://doi.org/10.7554/eLife.76870

Share this article

https://doi.org/10.7554/eLife.76870

Categories and tags

Research organism

Further reading

    1. Cancer Biology
    2. Cell Biology

    Changes in local mineral homeostasis facilitate the formation of benign and malignant testicular microcalcifications

    Ida Marie Boisen, Nadia Krarup Knudsen ... Martin Blomberg Jensen
    Research Article

    Testicular microcalcifications consist of hydroxyapatite and have been associated with an increased risk of testicular germ cell tumors (TGCTs) but are also found in benign cases such as loss-of-function variants in the phosphate transporter SLC34A2. Here, we show that fibroblast growth factor 23 (FGF23), a regulator of phosphate homeostasis, is expressed in testicular germ cell neoplasia in situ (GCNIS), embryonal carcinoma (EC), and human embryonic stem cells. FGF23 is not glycosylated in TGCTs and therefore cleaved into a C-terminal fragment which competitively antagonizes full-length FGF23. Here, Fgf23 knockout mice presented with marked calcifications in the epididymis, spermatogenic arrest, and focally germ cells expressing the osteoblast marker Osteocalcin (gene name: Bglap, protein name). Moreover, the frequent testicular microcalcifications in mice with no functional androgen receptor and lack of circulating gonadotropins are associated with lower Slc34a2 and higher Bglap/Slc34a1 (protein name: NPT2a) expression compared with wild-type mice. In accordance, human testicular specimens with microcalcifications also have lower SLC34A2 and a subpopulation of germ cells express phosphate transporter NPT2a, Osteocalcin, and RUNX2 highlighting aberrant local phosphate handling and expression of bone-specific proteins. Mineral disturbance in vitro using calcium or phosphate treatment induced deposition of calcium phosphate in a spermatogonial cell line and this effect was fully rescued by the mineralization inhibitor pyrophosphate. In conclusion, testicular microcalcifications arise secondary to local alterations in mineral homeostasis, which in combination with impaired Sertoli cell function and reduced levels of mineralization inhibitors due to high alkaline phosphatase activity in GCNIS and TGCTs facilitate osteogenic-like differentiation of testicular cells and deposition of hydroxyapatite.

    1. Cell Biology
    2. Genetics and Genomics

    Pyruvate and related energetic metabolites modulate resilience against high genetic risk for glaucoma

    Keva Li, Nicholas Tolman ... UK Biobank Eye and Vision Consortium
    Research Article

    A glaucoma polygenic risk score (PRS) can effectively identify disease risk, but some individuals with high PRS do not develop glaucoma. Factors contributing to this resilience remain unclear. Using 4,658 glaucoma cases and 113,040 controls in a cross-sectional study of the UK Biobank, we investigated whether plasma metabolites enhanced glaucoma prediction and if a metabolomic signature of resilience in high-genetic-risk individuals existed. Logistic regression models incorporating 168 NMR-based metabolites into PRS-based glaucoma assessments were developed, with multiple comparison corrections applied. While metabolites weakly predicted glaucoma (Area Under the Curve = 0.579), they offered marginal prediction improvement in PRS-only-based models (p=0.004). We identified a metabolomic signature associated with resilience in the top glaucoma PRS decile, with elevated glycolysis-related metabolites—lactate (p=8.8E-12), pyruvate (p=1.9E-10), and citrate (p=0.02)—linked to reduced glaucoma prevalence. These metabolites combined significantly modified the PRS-glaucoma relationship (Pinteraction = 0.011). Higher total resilience metabolite levels within the highest PRS quartile corresponded to lower glaucoma prevalence (Odds Ratiohighest vs. lowest total resilience metabolite quartile=0.71, 95% Confidence Interval = 0.64–0.80). As pyruvate is a foundational metabolite linking glycolysis to tricarboxylic acid cycle metabolism and ATP generation, we pursued experimental validation for this putative resilience biomarker in a human-relevant Mus musculus glaucoma model. Dietary pyruvate mitigated elevated intraocular pressure (p=0.002) and optic nerve damage (p<0.0003) in Lmx1bV265D mice. These findings highlight the protective role of pyruvate-related metabolism against glaucoma and suggest potential avenues for therapeutic intervention.