Modulation of Endocytosis by the Crumbs/BH-spectrin Complex

目的和方法：在多细胞动物体内，上皮细胞层形成外部世界之间和隔室之间的边界。上皮细胞的一个关键特征是分裂成顶区和基底侧区，由带状黏附复合体(如带状黏附复合体)分隔。这种细胞的不对称性或基底部的极性是由蛋白质复合体在上皮细胞中产生的，这些蛋白质复合体重组细胞内部，将不同组的蛋白质输送到顶端和基底外侧表面。在极化过程中，一个保守的蛋白质复合体诱导顶端区域的形成，并组织贴壁的带状结构。托马斯博士将以果蝇为模型系统，分析一组相互作用的蛋白质的作用，这些蛋白质参与了极性形成的下游步骤，这些步骤具体导致了小带粘连的稳定。这些蛋白质包括Crumbs、Annexin B9和Beta-Heavy Spectrin。初步实验表明，这些蛋白质形成一条调节蛋白质在贴壁带上周转速度的途径的假说成立，也可能决定顶端极性蛋白的水平，如Crumb。实验目的是测试β-Heavy Spectrin和Annexin B9的作用(S)及其合作：(I)设置顶端极性决定因素的平衡水平，以及对贴壁带中蛋白质水平的影响，以及(Ii)它们在维持膜面积方面的作用。作为这项工作的一部分，将直接测量在野生型和突变背景下DE-钙粘附素在交界处的周转率的差异。将使用一种利用果蝇遗传学力量的多学科方法，结合现代细胞生物学技术，对同一标本中的野生型和突变条件进行并列比较，以揭示这些细胞特性的数量变化。智力优势：上皮细胞组织代表后生动物身体计划中的一种基本组织类型，这种器官在它们的发育、结构和生理上起着至关重要的作用。为了成功地建立上皮，细胞必须形成根尖基底部的极性，这与细胞-细胞黏附复合体的发展密切相关。虽然科学家们已经开始了解顶极形成的起始，但诱导顶极的蛋白质复合体的稳定性以及相关的小带粘附物的稳定性仍然不是很清楚。这些实验将测试和定义血影蛋白、顶端极性复合体和局部蛋白质/膜周转率之间的关系，使用两种成熟的工具和一套新的探针，以产生对上皮组织维持的新见解，这应该广泛适用于我们对所有上皮细胞的理解。更广泛的影响：该项目将在科学教育领域产生更广泛的影响。该项目不仅将为研究生提供培训，还将继续促进托马斯博士致力于本科生的研究培训(3-5个学期)。这些学生不仅包括那些为荣誉论文做项目的学生，还包括通过他持续参与宾夕法尼亚州立大学的WISER(科学与工程研究中的女性)和MURE(少数民族本科生研究经验)项目的代表不足的群体。它还计划在一个大的(约350名学生)细胞生物学入门课程中试验“跟随”方法，该课程服务于几个专业。在这里，除了要求的实验练习外，托马斯博士还将介绍这些项目中一个更容易掌握的研究思路，被描述为课程的一个组成部分，并在整个学期的课堂上跟进，定期更新进展情况。这将有助于演示他们所了解的技术在实际生活中的应用，但不能作为实验部分的一部分进行介绍。与此同时，这将使一群人对基础研究的性质、速度和思维过程有更好的感受，其中许多人可能不会选择成为专业生物学家，而是应该成为消息灵通的公民。

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.