The development of the respiratory, lymphatic, excretory, and circulatory systems requires the assembly of cells into networks of highly branched tubes. How portions of a tubular network join into a continuous whole (anastomosis) is a poorly understood step in tubular morphogenesis. The proposed study of the structure and function of Drosophila Short Stop (Shot) provides a unique entry into the mechanism of anastomosis, initiates a genetic approach to dissecting this process, and addresses how actin and microtubules are coordinated during morphogenesis, a key issue in cell biology. In shot embryos, tracheal cells undergo normal branching morphogenesis, form a lumen within the segment, but fall to form a lumenal connection spanning the segment boundary. In wild-type embryos, tracheal cells accumulate actin on the side destined to form a lumen. In shot embryos, this polarization occurs in the center of the tracheal network, but not in cells that participate in anastomosis. The expression of a Shot A-GFP fusion in tracheal cells rescues these defects. Shot A contains an N-terminal actin binding domain whose activity may be regulated by the PIP2 phospholipid second messenger, a central rod domain predicted to be 200 nm long, and a C-terminal microtubule binding domain whose activity may be modified by Ca++. Thus, Shot's actin and microtubule binding domains are likely regulated by second messengers and spatially well-separated by a rod domain capable of spanning approximately 10% of the tracheal cell. shot's structure and morphogenetic phenotypes together suggest that the interactions between Shot and the cytoskeleton are dynamically controlled to effect morphogenesis on a 200 nm scale. shot is allelic to kakapo, a gene implicated in integrin-mediated cell adhesion, and has human homologs whose functions in development and disease are unknown. To understand the cell biology and biochemical mechanism of anastomosis, we will: l) Define the cytoskeletal changes and cells relevant to anastomosis. 2) Identify key domains in Shot A that are required for anastomosis. 3) Define the role of integrins in anastomosis. These studies will define how Shot effects morphogenesis, define the role of integrin signaling with respect to Shot activity, and will have broad relevance to understanding cell motility and morphogenesis. They will therefore enhance the treatment of abnormalities in angiogenesis and vascular development that occur in cancer and heart disease, and common birth defects such as inappropriate anastomosis of the trachea.
| Lee, Seungbok; Kolodziej, Peter A (2002) The plakin Short Stop and the RhoA GTPase are required for E-cadherin-dependent apical surface remodeling during tracheal tube fusion. Development 129:1509-20 |
