Graduate Student University of Pittsburgh Pittsburgh, Pennsylvania, United States
Natalie Hager (University of Pittsburgh)| Ceara McAtee (University of Pittsburgh)| Justina Warnick (University of Pittsburgh)| Christopher Szent-Gyorgyi (Carnegie Mellon University)| Marcel Bruchez (Carnegie Mellon University)| Adam Kwiatkowski (University of Pittsburgh)| Jeffrey Brodsky (University of Pittsburgh)| Allyson O'Donnell (University of Pittsburgh)
Recent advantages of genetically encoded fluorescent probes have led to the development of fluorogen-activating proteins (FAPs). This technology has two components: a non-fluorescent single chain antibody (SCA) that can be fused to a protein of interest and fluorogens, which in our case are derivatives of malachite green that are non-fluorescent when free in solution. When the SCA and fluorogen bind, there is a 20,000-fold fluorescent increase relative to unbound dye. The FAP-technology has two major advantages to standard fluorescent probes: i) the fluorogen exists as either a membrane-permeant or impermeant form and this allows for selective labeling of cell surface proteins and (2) since SCA does not fluoresce when it is not bound by dye and so there is no background fluorescence during the imaging of other markers until after the fluorogen is added. Although developed in yeast, this technology surprisingly has not been applied in this model system until our recent studies. In order to make this technology more available to the yeast community, we have optimized the SCA sequence for yeast expression, created a series for FAP-tagged plasmid constructs, and generated a suite of intracellular localization markers to aid in cell biology studies in yeast. We further employ the FAP technology to study the trafficking of the G-protein coupled receptor Ste3 and its regulation by the alpha-arrestins, an important class of trafficking adaptor.