IFIBYNE   05513
INSTITUTO DE FISIOLOGIA, BIOLOGIA MOLECULAR Y NEUROCIENCIAS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
DEVELOPMENT OF A MICROFLUIDIC-BASED METHOD FOR STUDING GRADIENT SENSING AT THE SINGLE CELL LEVEL
Autor/es:
VASEN G; BUSH A; BHATTACHARJEE N; FOLCH A; BRENT R; COLMAN-LERNER A
Reunión:
Congreso; L Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2014
Resumen:
gradient direction. We used the chemotropic growth towards mating pheromone (αFactor, αF) of S. cerevisiae as model system of gradient sensing. Here we present (1) the development of a reliable microfluidic-based method for analyzing chemotropism and (2) the generation of molecular tools for analyzing the dynamics of cell polarization. The microfluidic device used had open chambers to which two different media are delivered in opposite sides by a strip of microchannels. First, we determined the linearity of gradient, the time of formation and stability in time using fluorescent dyes. Next, we exposed cells with YFP under the control of an αF responsive promoter to different gradients of αF. We found an almost linear relation of position and reporter induction. When we measured the angle of the polarizations, we observed that cells determine direction more efficiently in gradients of 0-50nM. Once the method was established, we tagged Far1, Cdc24 and Bem1, key players in the polarization process, with a triple copy of mNeonGreen, a new bright and photostable fluorescent protein. Mutant strains showed normal αF response and the localization of fusion proteins could be tracked over time. With these method and tools we will pursue the study of gradient sensing in single cells.