• SELF-ASSOCIATION IS A COMMON PROPERTY IN BOVINE, MOUSE AND XENOPUSARRESTIN

      Calvert, Peter; Qiu, Shuang (2014)
      The light-driven translocation of arrestin from rod inner segment to outer segment was indicated to involve free diffusion with binding affinityto light-activated phosphorhodopsin, however, it is still debatable how arrestin is excluded from the dark-adapted outer segment. Previousstudies demonstrated that bovine visual arrestin had the property of self-association. The self-association propertyof both wild-type and mutated purified visual arrestin of several species (bovine, mouse and Xenopus laevis) was studiedby performing analytical ultracentrifugation experimentswhich providedthe oligomer formation information and association constants.Theself-association parameters of purified bovine and mousevisual arrestinwere investigated and compared with other studies. Results showed that arrestin of both species could self-associate, forming dimersin a concentration-dependent manner, but tetramerswere not detected at the highest concentrations examined.Xenopus arrestin was shown to self-associateas well, existing in a monomer-dimer equilibrium with the dimer dissociation constantKD,dim=80.8μM, which suggestedthat self-association was also a feature of Xenopus visual arrestin. Interestingly, homologous mutations (F78A/Y84A/F193A)of Xenopusarrestin, which were supposed to beconstitutive monomers, failed to completely disrupt the oligomerizationof Xenopusarrestinwith the dimer dissociation constantKD,dim=200.7μM , indicating that these regions were not conserved in amphibian visual arrestin, includingXenopuslaevis. The percentage of the dimer was higher than that of monomer at physiological concentrations in all species of arrestins tested.
    • STUDYING RETINA DEVELOPMENT USING SMALL MOLECULE BMP INHIBITORS AND MAKING STABLE MOUSE EMBRYONIC STEM CELL LINES

      Viczian, Andrea; Keshvani, Caezaan (2013)
      Age related macular degeneration results in loss ofconephotoreceptors. Studies have not been able to efficientlytransplant cone cells, possiblydue to their limited numbersand lack of information about their development in the mammalian retina.Our lab has discoveredthat BMP signal inhibition is important for eye developmentin the frogas well as generating retinal cone photoreceptorsin mouse embryonic stem cell cultures. The frog, Xenopus laevis,can grow eyeswithin a few days; it is also amenable to transplantation and genetic alteration. In this study, two small molecule BMP inhibitors, LDN193189 and Dorsomorphin, causedshortened tails(dorsalized) phenotypein Xenopusembryos as well asexpansionofthe neural plate in neural stage embryos. This suggests that these chemical inhibitors can be used in place of the standard BMP antagonist, Noggin, in cell culture experiments. Mouse embryonic stem cell cultures treated with Noggin have been used to generate retinal progenitors that generate cone photoreceptorsin our lab. In order to further study cones indetail,we needed to enrich the population of cone photoreceptors from retinal progenitors by marking these cells in-­‐vitro. We generated stable mouse embryonic stem cells expressing a promoter that drives expression in rod/cone progenitor cells upstream from the mCherry fluorescent protein. This construct also contained aubiquitous promoterdriving an antibiotic resistance genefor antibiotic selection. In future studies, these stable ES celllines could be used to differentiate into rod/cone progenitors selected specifically by FAC sorting. Small molecule BMP inhibitors could be used in place of Noggin and analyzed in the generation of conecells.