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Detecting nanoscale distribution of protein pairs by proximity dependent super-resolution microscopy

Alexander H. Clowsley, William T. Kaufhold, Tobias Lutz, Anna Meletiou, Lorenzo Di Michele, Christian Soeller
doi: https://doi.org/10.1101/591081
Alexander H. Clowsley
†Living Systems Institute & Biomedical Physics, University of Exeter, Exeter, UK
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William T. Kaufhold
‡Cavendish Laboratory, University of Cambridge, Cambridge, UK
§Department of Chemistry, Molecular Sciences Research Hub, Imperial College London, UK
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Tobias Lutz
†Living Systems Institute & Biomedical Physics, University of Exeter, Exeter, UK
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Anna Meletiou
†Living Systems Institute & Biomedical Physics, University of Exeter, Exeter, UK
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Lorenzo Di Michele
§Department of Chemistry, Molecular Sciences Research Hub, Imperial College London, UK
‡Cavendish Laboratory, University of Cambridge, Cambridge, UK
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Christian Soeller
†Living Systems Institute & Biomedical Physics, University of Exeter, Exeter, UK
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  • For correspondence: C.Soeller@exeter.ac.uk
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ABSTRACT

Interactions between biomolecules such as proteins underlie most cellular processes. It is crucial to visualize these molecular-interaction complexes directly within the cell, to show precisely where these interactions occur and thus improve our understanding of cellular regulation. Currently available proximity-sensitive assays for in-situ imaging of such interactions produce diffraction-limited signals and therefore preclude information on the nanometer-scale distribution of interaction complexes. By contrast, optical super-resolution imaging provides information about molecular distributions with nanometer resolution which has greatly advanced our understanding of cell biology. However, current co-localization analysis of super-resolution fluorescence imaging is prone to false positive signals as the detection of protein proximity is directly dependent on the local optical resolution. Here we present Proximity-Dependent PAINT (PD-PAINT), a method for sub-diffraction imaging of protein pairs, in which proximity detection is decoupled from optical resolution. Proximity is detected via the highly distance-dependent interaction of two DNA labels anchored to the target species. Labeled protein pairs are then imaged with high contrast and nanoscale resolution using the super-resolution approach of DNA-PAINT. The mechanisms underlying the new technique are analyzed by means of coarse-grained molecular simulations and experimentally demonstrated by imaging DNA-origami tiles and epitopes of cardiac proteins in isolated cardiomyocytes. We show that PD-PAINT can be straightforwardly integrated in a multiplexed super-resolution imaging protocol and benefits from advantages of DNA-based super-resolution localization microscopy, such as high specificity, high resolution and the ability to image quantitatively.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted May 30, 2020.
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Detecting nanoscale distribution of protein pairs by proximity dependent super-resolution microscopy
Alexander H. Clowsley, William T. Kaufhold, Tobias Lutz, Anna Meletiou, Lorenzo Di Michele, Christian Soeller
bioRxiv 591081; doi: https://doi.org/10.1101/591081
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Detecting nanoscale distribution of protein pairs by proximity dependent super-resolution microscopy
Alexander H. Clowsley, William T. Kaufhold, Tobias Lutz, Anna Meletiou, Lorenzo Di Michele, Christian Soeller
bioRxiv 591081; doi: https://doi.org/10.1101/591081

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