Versatile Super-Resolution Calibration Standard for Quantifying Protein Copy Number

Abstract

Single molecule based super-resolution microscopy offers a unique opportunity for quantifying protein copy numbers with nanoscale resolution [1,2]. While fluorescent proteins have been extensively characterized for quantitative imaging using calibration standards, similar calibration tools for small organic fluorophores used in conjunction with immunofluorescence are lacking.Within this framework, the development of methods able to access a precise molecular counting of protein copy numbers is essential, clearing the way to address several biological questions using super-resolution techniques such as stochastic optical reconstruction microscopy (STORM).The development of a suitable calibration method represents the best way to address the challenges of molecular counting using super-resolution [3,4]. Within this project, we demonstrate that DNA origami in combination with GFP antibodies is a versatile platform for quantifying protein copy number in immunofluorescence based super-resolution microscopy. We show that this calibration method, besides quantifying the average protein copy number in a cell, allows determining the abundance of various oligomeric states. Furthermore, we apply this calibration method to quantify nucleoporins (NUP107) [5] and molecular motors (dynein intermediate chain) [6] in vivo. Overall, we provide a versatile strategy for quantifying a large number of proteins of interest using various labeling approaches.1. Durisic, N., et al., Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate. Nat Methods, 2014. 11(2): p. 156-62.2. Ulbrich, M.H. and E.Y. Isacoff, Subunit counting in membrane-bound proteins. Nat Methods, 2007. 4(4): p. 319-21.3. Schmied, J.J., et al., DNA origami-based standards for quantitative fluorescence microscopy. Nat Protoc, 2014. 9(6): p. 1367-91.4. Jungmann R. et al. Quantitative superresolution imaging with qPAINT Nature methods doi:10.1038/nmeth.3804 (2016)5. Szymborska, A., et al., Nuclear Pore Scaffold Structure Analyzed by Super-Resolution Microscopy and Particle Averaging. Science, 2013. 341(6146): p. 655-658.6. Derr, N.D., et al., Tug-of-war in motor protein ensembles revealed with a programmable DNA origami scaffold. Science, 2012. 338(6107): p. 662-5.