Protein association dynamics studied by super resolution fluorescence microscopy

MORALES VÁSQUEZ, MIGUEL A.; EDUARDO ARTZ; LUCAS TEDESCO; PEDRO ARAMENDIA; ALAN SZALAI

Cordoba-Villa Carlos Paz

Congreso; XIII ELAFOT; 2017

Universidad Nacional de Córdoba

Fluorescence nanoscopy render molecular localization within a precision of tens of nanometers, 10-50 times lower than the diffraction limit. This possibility opens the way to study molecular colocalization or association between species in the intermediate range between FRET (1-10 nm) and conventional confocal microscopy (hundreds of nanometers), provided the target species can be distinguished by their fluorescent marker.The quantitative analysis of the colocalization distribution of two emitting species was approached in many ways: cluster density in color scale, cluster analysis, spatial correlation function and comparison with regular patterns.In this work, we analyze the application of distribution functions to quantify the extent of molecular association in one and two color Stochastic Optical Reconstruction Microscopy (STORM) image sets.From molecular localization maps we obtain the first neighbor distance (dFN) between molecules of the same (A-A and B-B) and of different species (A-B and B-A). We build the complementary cumulative distribution function (CCDF) of dFN in each case. The analysis and comparison of these four CCDF can distinguish between: 1) association and distribution in a two dimensional compartment, 2) a one dimensional environment (membrane or filament), 3) a 1:1 association or 4) a clustering of one type of molecules around the other.We apply this methodology to the analysis of the influence of RSUME (RWD-domain-containing sumoylation enhancer) on the dynamics of the HIF-VHL (Hypoxia-inducible factor, von Hippel Lindau) complex. To this aim, in a set of experiments carried out under identical conditions, we mark the proteins pairwise with two different rhodamine dyes by the postraductional modifications known as SNAP-CLIP. Localization is performed at the single molecule level by two color STORM experiments.We demonstrate that the dyes are adequate for these type of experiments and analyze the cellular distribution of proteins by comparing the actual distribution with simulated random and associated distributions performed in the same location pattern.