2009 USDA-supported Students
Raquel Castaneda, Universidad Metropolitana
Characterization of Directed Evolution Clones of the Fluorogen Activating Protein J6 and the Fluorogen Dimethylindole Red (DIR)
The fluorogen activating proteins (FAPs) are currently used in research to study cellular events in the cytoplasm and on the cell surface. These proteins are derived from single chain variable fragments, which consist of a variable heavy (VH) and variable light (VL) domains connected by a flexible linker. Different dyes interact with different FAPs to emit florescence. This florescence emitted from the particular fluorophore allows us to find the FAP. The fluorophore used in this specific research project was the fluorophore dimethylindole red (DIR). Previously, the J6-DIR FAP was mutated by error-prone PCR method. Clones were selected with increased binding affinity or fluorescence. This experiment produced different clones of the J6 FAP. The objective of this research was determine which amino acid changes in the mutant clones of the J6 FAP were responsible for the increase in the fluorescence. This information will allow us to determine which of these clones of FAP is the most efficient to obtain a greater amount of fluorescence at different concentrations of DIR. In my experiments, I used restriction enzymes and molecular biology techniques on the mutant J6 plasmids to separate the changed amino acids into individual plasmids. These plasmids were cultured in S. cerevisiae and grown to express the protein on the cell surface. DIR was added to these cultures at different concentrations (5 nM and 100 nM). This allowed us to observe by flow cytometry which clones carried changes that improved DIR binding or fluorescence. Of the sixteen single amino acid changes that were derived from the mutant clones of J6 FAP, two produced the most significant improvements in the binding or fluorescence activation of DIR. They were the VH mutation I58T, in clones E12, C8, B11, K10 and F8 and the VL mutation V212I, found in clones B15 and L5.
Joshua Millan, University of Puerto Rico in Aguadilla
Signaling Pathways to Tissue Morphogenesis
The creation of form during development is known as morphogenesis. For example, a type of tissue bending, invagination, is known to be involved in the neurulation of vertebrates and results in the bending of the dorsal neuroepithilium. Very little is known about the molecular pathways necessary for tissue morphogenesis. The purpose of this study is to determine how signaling pathways influence cytoskeletal reorganization and consequently cell shape change in tissue morphogenesis. We are examining fold formation in the developing Drosophila wing epithelium as a model system to identify the proteins and pathways required for this morphogenetic event. Previous work in the lab examining the apical domains of cells in the developing folds revealed that the originally hexagonal cells transform into rectangles elongated along the anterior/posterior axis. Studies of epithelial morphogenesis in Drosophila and other organisms have established a relationship between epithelial tissue folding and Myosin II activity. Therefore, we have started testing whether Myosin II and the Rho signaling pathway, a known activator of Myosin II, play a role in tissue folding in the larval wing epithelium. Using the FLP-out and UAS/GAL4 systems we expressed Sqh-AA, a dominant negative form of the Myosin regulatory light chain Sqh, in patches of wing epithelial cells. We found that this resulted in weak effects on cell elongation and apical constriction, without appearing to affect the folding of the epithelium. This may indicate that Myosin II is not necessary for tissue folding in the Drosophila imaginal disc. Alternatively disruption of Myosin II with Sqh-AA may not be sufficient to disrupt fold formation. We predict that a more significant disruption of Rho signaling or Myosin II will result in disruption of imaginal disc folding. These experiments will provide insight into the molecular pathways that underlie morphogenesis during development.