This proposal is for the acquisition of a Drosophila chamber to replace an old and broken chamber, which was used to culture Drosophila strains in our laboratory. The awarded NIH R15 research project is to study the role of a poorly understood Osiris (Osi) gene family in regulating tube maturation in Drosophila trachea, which is a premier system to reveal fundamental mechanisms of tubular organ formation. Malformation of tubes leads to various human diseases, such as polycystic kidney disease and vascular diseases. The Drosophila trachea is a ramifying network of epithelial tubes with a monolayer of epithelial cells surrounding an apical lumen. During tube maturation, the apical secretion burst deposits large amounts of luminal matrix components to the apical extracellular lumen. Then, this lumen is cleared before air can fill the lumen. Previous studies have shown that components of protein trafficking pathways have been implicated in the secretion/clearance of luminal proteins. The objective of the awarded project is to reveal the functions of Osi proteins as “broader coordinators” of protein trafficking during tube maturation. To test this hypothesis, we will complete the following three specific aims: Aim. 1 Determine the function of Osi genes in trafficking of apical luminal matrix during tube maturation. Aim. 2: Determine the function of Osi genes as coordinators to control trafficking components. Aim. 3: Identify proteins that directly bind to Osi proteins. To fulfill these goals, we will generate fly strains that carrying endosome markers, luminal protein markers in Osi mutant background to determine changes of endosomes and luminal proteins in Osi mutants. Then, we will generate lines to express Osi transgenes or endosome transgenes in Osi mutants to analyze the rescue effect. Thereafter, we will generate fly strains carrying individual endosome mutations in Osi mutants to test genetic interaction between Osi genes and endosome genes. Finally, we will express HA-tagged Osi genes in trachea to isolate proteins that interact with Osi proteins. Therefore, to carry out the proposed research, fly strains carrying multiple mutations, balancers, and transgenes have been and will be generated. Currently the fly strains are cultured at room temperature in the lab. Unfortunately dozens of strains that were generated for the project were lost due to unexpected significant decrease or increase of the temperatures. The proposed instrument, a fly chamber, Model DR-36VL, from GENEVA SCIENTIFIC maintains proper humidity, light cycles, and stable temperatures will keep the fly strains not only alive but thriving. Without it, it will not be possible to carry out the proposed experiments in a timely manner.