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

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 obtaining 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 colocalization 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. Labelled 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 high proximity biotin binding sites of streptavidin and epitopes of ryanodine receptor proteins in cardiac tissue samples. We show that PD-PAINT can be straightforwardly integrated in a multiplexed superresolution 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.Introduction. Characterizing protein interactions by detection of protein-protein complexes is the basis of understanding many processes in biology 1 . Often, these are detected by in vitro methods such as co-immunoprecipitation, cross-linking or affinity blotting 2,3 . It is increasingly evident that besides detecting the mere presence of protein-protein interactions, it is important to determine where these occur within a cell or tissue, since the nanoscale organization of signaling complexes directly controls cell function 4,5 . To this end, methods have been developed that are based on labelling the features of interest with synthetic DNA oligonucleotides, conjugated to antibodies or other molecular markers. The oligonucleotides act as proximity probes, and a subsequent amplification step is implemented to produce a fluorescent signal detectable by a conventional microscope. In an (in situ) proximity ligation assay (PLA), enzymatic amplification occurs via the rolling circle method 5-8 , while in the ProxHCR scheme amplification is non-enzymatic and relies on a hybridization chain reaction 9,10 . However, the high fluorescent amplification in both methods also ef...

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