Understanding the integration of a protein in a complex network: “the living cell

GOMEZ GA

Oro Verde, Argentina

Workshop; II Workshop de microscopía tridimensional; 2007

Facultad de Ingeniería Universidad Nacional de Entre Ríos

Protein functions are regulated in a multivariable network, which results of the integration of protein transport, postranslational modifications and specific interactions, which ocurrs in diferents subcelular compartments at diferent time scales. Integrate the knowledge about the function of a protein (or a lipid), implicate to know all these aspects. Fluorescence microscopy has emerged as an appropiate tool to investigate them, which is a relevant complement for biochemical and genetical approachs. In particular, confocal microscopy, which will be treated by other speakears in the present workshop, allow to determine the spatial distribution of the expresed protein. In the present talk, we will focus in the study of protein dynamics and protein interactions in the living cell. Protein dynamics, would be studied by two methodologies, FRAP (Fluorescence Recovery After Photobleaching) and FCS (Fluorescence Correlation Spectroscopy). FRAP methodologies allow to describe dynamics at three diferent levels of organization. FRAP experiments would be applied to study protein diffusion inside diferents compartments, protein-membrane association, lateral protein segregation and protein exchange between diferents reservoirs, all of them ocurring at diferent time scales. FCS, is aimed to the single molecule level, and allow to determine, intrinsic diffusion constants and molecular association, which are parameters that would be applied to model complex recovery curves obtained in FRAP experiments. Other techniques, to study protein dynamics at the molecular level, such as SPT (Single Particle Tracking), which will be also discussed. The remaining question is about the interaction network around a protein. FRET (Fluorescence Resonance Energy Transfer) microscopy is utilized to assort proximity between two proteins. Because of high dependence of FRET on distance, such technique is applied in two contexts. Using a FRET intermolecular approach, we could determine specific protein-protein interactions or lateral protein segregation in membranes and where these ocurr inside the cell, while using a FRET intramolecular approach, we can determine molecular rearragments in protein conformation, which can result of ligand binding or protein association. FRET intramolecular is appropiate to construct specific probes to study molecular activation. After discuss both groups of methodologies, FRAP and FRET, and their limitations, we could integrate the three data in a general context and finally, ...Can we understand how proteins work in a complex network?Supported by: F. Antorchas, FONCYT, CONICET, SECyT-UNC