• FUNCTIONAL CHARACTERIZATION OF DENND5A/RAB6IP1 IN EARLY EMBRYONIC DEVELOPMENT

      Habas, Raymond; Amini, Shohreh (Temple University. Libraries, 2015)
      Denn stands for differentially expressed in normal vs neoplastic cells, Dennd5a or Denn domain containing protein 5a is also named Rab6 interacting protein 1 (Rab6ip1), KIAA1091 (HUGE database). Rab6 is a small GTP-binding protein that involves in intra-Golgi transport. In a yeast two hybrid screen by Bruno Goud group in 1995, they used Rab6 as bait and found several interacting cDNA clones including Rab6 interacting protein 1 or Dennd5a. Several Dennd5a interaction partners have been discovered, Rab6, Rab11 and Sorting Nexin 1, which primarily locate Dennd5a to intracellular transport pathway. In Dr. Habas lab, Dennd5a was identified as a Daam1 interacting protein in a yeast two hybrid screen. Daam1 is a key player in non-canonical Wnt signaling and regulates vertebrate gastrulation and neural tube closure. I confirmed that Daam1 can physically bind to Dennd5a in immunoprecipitation assays using epitope tagged constructs. In the cellular level experiment, Dennd5a can colocalize with Daam1 on/near cell membrane and within punctate structures, however, Wnt5a and Wnt3a stimulations don't appear to affect their subcellular colocalization. In Xenopus in-vivo experiments, knockdown of Dennd5a by morpholino leads to unclosed blastopore and “open-back” defect during embryonic development. Interestingly, overexpression of Dennd5a by microinjection of RNA leads to very similar phenotype.