Recycling: An Alternative Method for Rapid Gap Junction Plaque Assembly

间隙连接是细胞间通讯的关键通道。间隙连接的主要成分是连接蛋白。 本项目旨在描述缝隙连接在细胞表面组装的新机制。 该项目将确定连接蛋白在形成新的功能性间隙连接通道中可回收和再利用的程度。它还将确定调节连接蛋白再循环与降解的分子机制。 总之，这将提供一个更好的理解的营业额和贩运的连接蛋白。这些知识将有助于理解细胞间通讯至关重要的许多细胞现象。实验收集的数据也将用于开发软件，该软件将在数学上预测连接蛋白的修饰如何决定其生物学功能，包括移动到不同的细胞质区室。该项目的更广泛的影响是三方面的，扩大科学参与，创造新的科学导向的艺术展览，并提供研究培训。将通过为匹兹堡大学、匹兹堡周边广大农村地区的小型学院和少数民族服务机构的个人提供机会，使他们有机会参与创新研究，从而加强对科学的参与。 将向各级研究人员提供培训，从本科生和研究生到博士后研究员和客座教授。 他们将获得使用尖端成像和数学建模工具的培训，以研究蛋白质动力学，回收和降解。 科学和计算研讨会，学生将积极使用建模软件来分析实验形态数据，将导致新的见解，以及有趣的培训和职业道路。此外，学生将被引导在装置艺术描绘蛋白质动力学和调节细胞行为的生产。这些艺术展览将在讲习班、社区和当地艺术博物馆展出。这些展览旨在鼓励科学，技术，工程和数学（STEM）的职业道路。此外，他们还将向公众介绍和熟悉研究世界，突出活细胞的美丽和复杂性。间隙连接的主要蛋白亚基是连接蛋白，其中最丰富的是连接蛋白43。该项目将测试连接蛋白43的再循环有助于形成新的间隙连接通道的假设。 具体地说，本提案的第一个目的将确定连接蛋白43在多种细胞类型中内吞作用后的动力学。 在第二个目标中，假设将被测试，内吞和回收的连接蛋白可以调节多种激酶和磷酸酶。为了检验这些假设，PI将使用生物化学、共聚焦显微镜、形态学和细胞生物学方法，并结合磷模拟物和药理学激活剂。 目标3将使用内吞、再循环和降解的动力学参数来生成微分速率方程模型，该模型将表征和预测连接蛋白运输的细节。 从这项研究中获得的信息和提供的培训将促进细胞间通讯科学的发展，这是理解复杂生理过程的最终需要，从而有助于公共福利。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估来支持。

Gap junctions are cellular channels that are critical for cell-to-cell communication. The main component of gap junctions are the connexin proteins. This Project is expected to describe a new mechanism by which gap junctions assemble at the surface of the cell. This Project will determine the extent that connexins can be recycled and re-used in the formation of new functional gap junction channels. It will also identify the molecular mechanisms that regulate connexin recycling versus degradation. Together, this will provide a better understanding of the turnover and trafficking of connexins. This knowledge will be useful for understanding the numerous cellular phenomena in which cell-cell communication is critical. Experimentally collected data will also be used to develop software that will mathematically predict how the modification of connexins determines their biological functions, including movement to different cytoplasmic compartments. The Broader Impacts of the Project are three-fold, broadening participation in science, creating novel science-oriented art exhibits, and providing research training. Participation in science will be strengthened by providing an opportunity for individuals from the University of Pittsburgh, smaller colleges from extended rural areas surrounding Pittsburgh, and minority serving institutions to participate in innovative research opportunities, to which they would not otherwise have access. Training will be provided to researchers at all levels, from undergraduates and graduate students to postdoctoral fellows and visiting professors. They will gain hands on training in the use of cutting edge imaging and mathematical modeling tools to study protein dynamics, recycling and degradation. Scientific and computational workshops, where students will actively use modeling software to analyze experimental morphological data, will lead to novel insights, as well as interesting training and career paths. In addition, students will be directed in the production of installation art depicting protein dynamics and regulated cell behavior. These art exhibits will be presented at workshops, in the community, and at local art museums. These exhibits are intended to encourage career paths in science, technology, engineering, and math (STEM). In addition, they will introduce and familiarize the public with the world of research, highlighting the beauty and intricacy of living cells. The major protein subunits of gap junctions are the connexin proteins, the most abundant of which is connexin 43. The Project will test the hypothesis that recycling of connexin 43 contributes to the formation of new gap junction channels. Specifically, the first Aim of this proposal will determine the dynamics of connexin 43 after endocytosis in multiple cell types. In the second Aim, the hypothesis will be tested that endocytosis and recycling of connexins can be regulated by multiple kinases and phosphatases. To test these hypotheses, the PI will use biochemistry, confocal microscopy, morphological, and cell biological methods, in combination with phosphor-mimetics and pharmacological activators. Aim 3 will use the dynamic parameters for endocytosis, recycling and degradation to generate a differential rate-equation model that will characterize and predict details of connexin trafficking. Information gained from this study and the training provided will advance the science of cell-cell communication that is ultimately needed to understand complex physiological processes, and thus contribute to public welfare.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.