INVESTIGADORES
ARAMENDIA Pedro Francisco
congresos y reuniones científicas
Título:
Protein association dynamics studied by super resolution fluorescence microscopy
Autor/es:
MIGUEL ANGEL MORALES VÁZQUEZ; LUCAS TEDESCO; ALAN SZALAI; EDUARDO ARZT; PEDRO F. ARAMENDÍA
Lugar:
Carlos Paz
Reunión:
Congreso; XIII ENCUENTRO LATINOAMERICANO DE FOTOQUIMICA Y FOTOBIOLOGIA; 2017
Resumen:
p { margin-bottom: 0.25cm; direction: ltr; color: rgb(0, 0, 0); line-height: 120%; }p.western { font-family: "Times New Roman",serif; font-size: 12pt; }p.cjk { font-family: "MS Mincho","MS 明朝",monospace; font-size: 12pt; }p.ctl { font-family: "Times New Roman",serif; font-size: 12pt; }a:link { color: rgb(0, 0, 255); }Fluorescence nanoscopy rendermolecular localization within a precision of tens of nanometers,10-50 times lower than the diffraction limit. This possibility opensthe way to study molecular co localization or association betweenspecies in the intermediate range between FRET (1-10 nm) andconventional confocal microscopy (hundreds of nanometers), providedthe target species can be distinguished by their fluorescent marker.The quantitative analysis ofthe colocalization distribution of two emitting species wasapproached in many ways: cluster density in color scale, clusteranalysis, spatial correlation function and comparison with regularpatterns.Inthis work, we analyze the application of distribution functions toquantify the extent of molecular association in one and two colorStochastic Optical Reconstruction Microscopy (STORM) image sets. Frommolecular 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 distributionfunction (CCDF) of dFNin each case. The analysis and comparison of these four CCDF candistinguish between: 1) association and distribution in a twodimensional compartment, 2) a one dimensional environment (membraneor filament), 3) a 1:1 association or 4) a clustering of one type ofmolecules around the other.We apply this methodology tothe analysis of the influence of RSUME (RWD-domain-containingsumoylation 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, wemark the proteins pairwise with two different rhodamine dyes by thepostraductional modifications known as SNAP-CLIP. Localization isperformed at the single molecule level by two-color STORMexperiments.We demonstrate that the dyesare adequate for these type of experiments and analyze the cellulardistribution of proteins by comparing the actual distribution withsimulated random and associated distributions performed in the samelocation pattern.
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