Dynamic Modulation of Protein Recycling by Spectrin

智力上的优点。上皮细胞是所有多细胞动物身体结构中必不可少的组织类型，在发育、结构和生理方面起着至关重要的作用。上皮细胞也是人体内最常见的组织类型。为了成功地建立上皮，细胞必须形成“极性”--细胞蛋白质分布的强烈不对称性。许多上皮功能，从营养吸收到器官形成，都依赖于蛋白质的不对称分布和上皮的这一基本属性。极性是由重组细胞的蛋白质组产生的，这些蛋白质组将不同组的蛋白质输送到每个主要的上皮表面：顶端、侧端和底部。在这个过程中，特定的蛋白质组负责特定顶面的形成。这个项目将以果蝇(DroSingila Blackogaster)为模型系统，分析一组蛋白质在导致顶端表面蛋白质稳定的步骤中所起的作用。这些蛋白质包括膜相关蛋白Annexin B9和βH Spectrin。初步数据表明，这种膜联蛋白和血影蛋白形成了一种复合体，决定了顶膜上蛋白质的水平，它们是通过调节细胞内蛋白质在被称为“蛋白质循环”的过程中进出顶端区域的运动来实现这一点的。为了验证这一假设，该项目将定量测量这两种蛋白质对正常和突变细胞和组织中蛋白质循环的影响。在这一过程中，Annexin B9和βH Spectrin还调节一种称为Rac的主要极性调节因子，这种关系也是研究的重点。最后，这一假说表明，许多许多蛋白质都可能以这种方式进行调节。因此，该项目还将开发一种方法，使用“蛋白质组”技术来识别所有这些蛋白质。虽然启动极化细胞表型的关键蛋白的作用已经开始被理解，但对初始顶端区域的稳定和细化仍然知之甚少。因此，该项目将对上皮组织的发展和维护产生新的见解。该项目还将有助于重新定义幽灵蛋白的总体作用。二十多年来，这些蛋白质在每一本基础细胞生物学教科书中都被描述为静态结构蛋白质。这项研究是越来越多的证据中的一部分，这些证据表明，这些蛋白质实际上在细胞中具有更广泛和更动态的作用，这对这一长期持有的观点提出了挑战。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和博士后研究员提供培训，还将把本科生主导的实验与研究相结合。本科生的参与者将从那些进行这些荣誉项目的人中挑选出来，也将从宾夕法尼亚州立大学的WISER(科学与工程研究中的女性)和Mure(少数族裔本科生研究经验)项目中选出代表不足的群体。此外，实验室实验将用于一个大型(500名学生)细胞生物学入门课程中的“现实科学”方法，该课程服务于几个专业。对于这种方法，由本科生研究人员执行的易于处理的研究思路随后被整合到课程(包括考试)中。这位本科生的实验在整个学期都会在课堂上进行，并定期更新学生的进步情况。本课程将演示课堂上提到但本课程实验部分无法涵盖的技术的实际应用。与此同时，这将使学生对基础研究的性质、速度和思维过程产生一种感觉，这些学生可能选择不成为专业生物学家，但仍将成为有科学素养的公民。

Intellectual merit. Epithelia are an essential tissue type in the body plan of all multicellular animals where they play a vital role in development, structure and physiology. Epithelia are also the most common tissue type in the body. To establish an epithelium successfully, cells must develop "polarity"--a strong asymmetry in the distribution of cellular proteins. Many epithelial functions, from nutrient uptake to organ formation, depend on such asymmetric distributions of proteins and this fundamental property of an epithelium. Polarity is generated by groups of proteins that reorganize the cell to transport different sets of proteins to each of the main epithelial surfaces: apical, lateral and basal. During this process, specific groups of proteins are tasked with the formation of the apical surface specifically. This project will analyze the role of a group of proteins involved in steps that lead to the stabilization of proteins at the apical surface using the fruit fly (Drosophila melanogaster) as a model system. These proteins include the membrane-associated proteins annexin B9 and beta H spectrin. Preliminary data has led to the hypothesis that this annexin and spectrin form a complex that determines the level of proteins at the apical membrane, and that they do this by modulating the movement of proteins inside the cell to and from the apical domain during a process called "protein recycling". To test this hypothesis the project will quantitatively measure the influence of these two proteins on protein recycling in normal and mutant cells and tissues. Annexin B9 and beta H spectrin also modulate a major polarity regulator called Rac during this process, and this relationship is also a focus of the research. Finally, the hypothesis suggests that many many proteins are likely to be modulated in this way. Therefore, the project will also develop a way to identify all such proteins using "proteomic" techniques. Although the action of the key proteins that initiate a polarized cell phenotype are beginning to be understood, the stabilization and elaboration of the incipient apical domain are still poorly understood. Thus, the project will produce new insights into the development and maintenance of epithelial organization. This project will also help redefine the role of spectrins in general. These proteins have been depicted in every basic cell biology textbook for over two decades as static structural proteins. This research is part of a growing body of evidence that demonstrates that these proteins actually have a much wider and more dynamic role in the cell that challenges this long-held view.Broader impacts. The broader impacts of this project will be in the area of science education. Not only will the project provide training for graduate students and postdoctoral fellows, but it will also integrate undergraduate-driven experiments with the research. Undergraduate participants will be drawn from those doing honors theses projects and also from underrepresented groups through the WISER (Women In Science and Engineering Research) and MURE (Minority Undergraduate Research Experience) programs at Penn State. In addition, lab experiments will be used for a "Reality Science" approach in a large (500 student) introductory cell biology class, which serves several majors. For this approach a tractable research thread that is performed by an undergraduate researcher is then integrated into the curriculum (including examinations). The undergraduate's experiments are followed in class throughout the semester with regular updates on that student's progress. This will demonstrate real-life applications of techniques that are mentioned in class, but which cannot be covered in the lab sections of the course. At the same time this will impart a feeling for the nature, pace, and thought processes that underly basic research amongst students who may choose not to become professional biologists, but nevertheless will become science-literate citizens.