CYTOSKELETAL EFFECTORS OF ANASTOMOSIS

吻合的细胞骨架效应器

• 批准号: 6286510
• 负责人: PETER A. KOLODZIEJ
• 金额: $ 18.22万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-01 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6286510
• 关键词: Drosophilidae; actins; binding proteins; cell adhesion; gene expression; gene mutation; histogenesis; integrins; invertebrate embryology; microtubules; protein isoforms; protein structure function; trachea

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.

呼吸道，淋巴，排泄和循环系统的发展需要将细胞组装到高度分支管的网络中。管状网络的一部分如何加入连续的整体（吻合）是肾小管形态发生的一步。 对果蝇短停止（SHOT）的结构和功能的拟议研究提供了独特的吻合机制，启动了一种遗传方法来剖析这一过程，并解决了肌动蛋白和微管在形态发生过程中如何协调，这是细胞生物学的关键问题。 在射击胚胎中，气管细胞经历正常的分支形态发生，形成了段内的腔，但落下以形成跨越段边界的流明连接。 在野生型胚胎中，气管细胞在注定形成管腔的侧面积聚肌动蛋白。 在射击胚胎中，这种极化发生在气管网络的中心，但不在参与吻合的细胞中。 气管细胞中SHOT A-GFP融合的表达挽救了这些缺陷。 Shot A包含一个N末端肌动蛋白结合结构域，其活性可以由PIP2磷脂第二信使（预测为200 nm）的中心杆域，并且C-末端微管结合结构域可通过Ca ++修饰。 因此，Shot的肌动蛋白和微管结合结构域可能受第二使者的调节，并在空间上由能够跨越大约10％的气管细胞的棒域进行了良好的分离。 Shot的结构和形态发生表型一起表明，射击与细胞骨架之间的相互作用受到动态控制，以在200 nm量表上作用形态发生。 Shot与Kakapo相位，该基因与整联蛋白介导的细胞粘附有关，并且具有人类同源物，其在发育和疾病中的作用尚不清楚。 为了了解吻合术的细胞生物学和生化机制，我们将：l）定义与吻合术相关的细胞骨架变化和细胞。 2）识别吻合所需的镜头A中的关键域。 3）定义整联蛋白在吻合中的作用。这些研究将定义射击效应形态发生，定义整联蛋白信号在射击活性方面的作用，并与理解细胞运动和形态发生具有广泛的相关性。 因此，它们将增强在癌症和心脏病中发生的血管生成和血管发育异常的治疗，以及常见的先天缺陷，例如气管的不适当吻合术。