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

  • 7,612
    views
  • 1,279
    downloads
  • 23
    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. Cell Biology

    High-throughput expansion microscopy enables scalable super-resolution imaging

    John H Day, Catherine M Della Santina ... Laurie A Boyer
    Tools and Resources

    Expansion microscopy (ExM) enables nanoscale imaging using a standard confocal microscope through the physical, isotropic expansion of fixed immunolabeled specimens. ExM is widely employed to image proteins, nucleic acids, and lipid membranes in single cells; however, current methods limit the number of samples that can be processed simultaneously. We developed High-throughput Expansion Microscopy (HiExM), a robust platform that enables expansion microscopy of cells cultured in a standard 96-well plate. Our method enables ~4.2 x expansion of cells within individual wells, across multiple wells, and between plates. We also demonstrate that HiExM can be combined with high-throughput confocal imaging platforms to greatly improve the ease and scalability of image acquisition. As an example, we analyzed the effects of doxorubicin, a known cardiotoxic agent, on human cardiomyocytes (CMs) as measured by the Hoechst signal across the nucleus. We show a dose-dependent effect on nuclear DNA that is not observed in unexpanded CMs, suggesting that HiExM improves the detection of cellular phenotypes in response to drug treatment. Our method broadens the application of ExM as a tool for scalable super-resolution imaging in biological research applications.

    1. Cell Biology
    2. Developmental Biology

    The exocyst complex controls multiple events in the pathway of regulated exocytosis

    Sofía Suárez Freire, Sebastián Perez-Pandolfo ... Mariana Melani
    Research Article

    Eukaryotic cells depend on exocytosis to direct intracellularly synthesized material toward the extracellular space or the plasma membrane, so exocytosis constitutes a basic function for cellular homeostasis and communication between cells. The secretory pathway includes biogenesis of secretory granules (SGs), their maturation and fusion with the plasma membrane (exocytosis), resulting in release of SG content to the extracellular space. The larval salivary gland of Drosophila melanogaster is an excellent model for studying exocytosis. This gland synthesizes mucins that are packaged in SGs that sprout from the trans-Golgi network and then undergo a maturation process that involves homotypic fusion, condensation, and acidification. Finally, mature SGs are directed to the apical domain of the plasma membrane with which they fuse, releasing their content into the gland lumen. The exocyst is a hetero-octameric complex that participates in tethering of vesicles to the plasma membrane during constitutive exocytosis. By precise temperature-dependent gradual activation of the Gal4-UAS expression system, we have induced different levels of silencing of exocyst complex subunits, and identified three temporarily distinctive steps of the regulated exocytic pathway where the exocyst is critically required: SG biogenesis, SG maturation, and SG exocytosis. Our results shed light on previously unidentified functions of the exocyst along the exocytic pathway. We propose that the exocyst acts as a general tethering factor in various steps of this cellular process.