Modulation of Endocytosis by the Crumbs/BH-spectrin Complex

目的和方法：上皮细胞层形成外部世界之间的边界和多细胞动物的隔室之间。上皮细胞的一个关键特征是它们被带状粘附复合物（如粘附小带）分隔成顶端和基底外侧域。这种细胞不对称性或顶基极性是由蛋白质复合物在上皮中产生的，这些蛋白质复合物重组细胞内部以将不同的蛋白质组递送到顶侧和基底侧表面。在极化过程中，一个保守的蛋白质复合物诱导顶端结构域的形成，并组织粘附小带。托马斯博士将分析一组相互作用的蛋白质的作用，这些蛋白质参与极性建立的下游步骤，这些步骤专门导致粘附小带的稳定，使用果蝇作为模型系统。这些蛋白质包括Crumbs，Annexin B 9和Beta-Heavy Spectrin。初步实验提出了这样的假设，即这些蛋白质形成了一条调节粘附小带蛋白质周转率的途径，实验目的是测试β-重血影蛋白和膜联蛋白B 9的作用以及它们对（i）设置的平衡水平的顶端极性决定因素和蛋白质水平的小带adherens，和（ii）他们的作用，在维持膜面积。作为这项工作的一部分，将直接测量DE-钙粘蛋白在野生型和突变背景的交界处的周转率的差异。一个多学科的方法，利用果蝇遗传学的力量将被使用，结合当代细胞生物学技术，在同一标本中进行野生型和突变体条件的并排比较，以揭示这些细胞特性的定量变化。智力优点：上皮细胞组织代表后生动物身体计划中的基本组织类型，并且这些器官在其发育、结构和生理学中起着至关重要的作用。为了成功地建立上皮，细胞必须形成顶基极性，这与细胞-细胞粘附复合物的形成密切相关。虽然科学家们开始了解顶极形成的起始，但诱导顶极的蛋白质复合物的稳定性以及相关粘附小带的稳定性仍然没有很好地理解。这些实验将测试和定义血影蛋白，顶端极性复合物和当地的蛋白质/膜周转率之间的关系，使用两个发达的工具，和一套新的探针，以产生新的见解上皮组织的维护，应广泛适用于我们的理解所有epithelias.Broader影响：更广泛的影响，这个项目将在科学教育领域。该项目不仅将为研究生提供培训，而且将继续促进托马斯博士对本科生研究培训的承诺（3-5/学期）。这些学生不仅包括那些对荣誉论文做项目，但也包括通过他在WISER（妇女在科学和工程研究）和MURE（少数民族本科生研究经验）在宾夕法尼亚州立大学计划持续参与代表性不足的群体。它还计划在一个大型（约350名学生）的细胞生物学入门课程中尝试“跟随沿着”的方法，该课程为几个专业提供服务。在这里，除了必要的实验练习，托马斯博士将介绍一个更容易处理的研究线索，从项目描述为课程的一个组成部分，并在整个学期的课堂上跟进，定期更新其进展情况。这将有助于展示他们所学习的技术的实际应用，但这些技术不能作为实验部分的一部分。与此同时，这将使一个群体对基础研究的性质、速度和思维过程有更好的感觉，他们中的许多人，也许是大多数人，不会选择成为专业的生物学家，但应该成为更知情的公民。

Objective and Methods: Epithelial cell layers form boundaries between the outside world and between compartments within multicellular animals. A key feature of epithelial cells is their division into apical and basolateral domains separated by belt-like adhesion complexes such as the zonula adherens. This cellular asymmetry, or apicobasal polarity is generated in epithelia by complexes of proteins that reorganize the cell interior to deliver different sets of proteins to the apical and basolateral surface. During polarization, a conserved protein complex induces the formation of the apical domain and organizes the zonula adherens. Dr. Thomas will analyze the role of a group of interacting proteins involved in steps immediately downstream of polarity establishment that lead specifically to the stabilization of the zonula adherens using Drosophila as a model system. These proteins include Crumbs, Annexin B9, and Beta-Heavy spectrin. Preliminary experiments suggest the hypothesis that these proteins form a pathway that regulates the rate of protein turnover at the zonula adherens, and may also set the level of apical polarity proteins such as Crumbs.The experimental objectives are to test the role(s) of Beta-Heavy spectrin and Annexin B9 and their collaboration to (i) set the equilibrium levels of the apical polarity determinants and upon protein levels in the zonula adherens, and (ii) their role in maintaining membrane area. As part of this effort, there will be direct measurement of differences in the turnover rate of DE-cadherin at the junction in wild-type and mutant backgrounds. A multidisciplinary approach that harnesses the power of Drosophila genetics will be used, combined with contemporary cell biology techniques, to perform side-by-side comparisons of wild-type and mutant conditions in the same specimen to reveal quantitative changes in these cellular properties. Intellectual merit: The epithelial cell organization represents an essential tissue type in the metazoan body plan, and such organs play a vital role in their development, structure and physiology. To successfully establish an epithelium, the cells must develop apicobasal polarity and this is intimately tied to the development of cell-cell adhesion complexes. Although scientists are beginning to understand the initiation of apical pole formation, the stabilization of the protein complexes that induce the apical pole, and the stabilization of the associated zonula adherens are still not well understood. These experiments will test and define the relationship between spectrin, the apical polarity complex and local rates of protein/membrane turnover using both well-developed tools, and a suite of novel probes to produce new insights into the maintenance of epithelial organization that should be broadly applicable to our understanding of all epithelia.Broader impacts: Broader impacts of this project will be in the area of science education. Not only will the project provide training for graduate students, but it will continue to facilitate Dr. Thomas's commitment to research training for undergraduates (3-5/semester). These students not only include those doing projects towards honors theses, but also include underrepresented groups through his on-going participation in the WISER (Women In Science and Engineering Research) and MURE (Minority Undergraduate Research Experience) programs at Penn State. It is also planned to experiment with a 'follow along' approach in a large (~350 student) introductory Cell Biology class that serves several majors. Here, in addition to required lab exercises, Dr. Thomas will introduce one of the more tractable research threads from the projects described as an integrated part of the curriculum, and follow up in class throughout the semester with regular updates on its progress. This will help demonstrate real-life applications of techniques that they learn about, but that cannot be covered as part of the lab sections. At the same time this will impart a better feeling for the nature, pace and thought processes underlying basic research amongst a group many of whom, perhaps most, will not choose to become professional biologists, but should become better-informed citizens.