Recycling: An Alternative Method for Rapid Gap Junction Plaque Assembly

回收：快速间隙连接斑块组装的替代方法

• 批准号: 2011577
• 负责人: Sandra Murray
• 金额: $ 87.73万
• 依托单位: University of Pittsburgh
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-15 至 2025-05-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2011577&HistoricalAwards=false
• 关键词: Recycling; Alternative; Method; Rapid; Gap

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.

缝隙连接是细胞间通讯的关键通道。缝隙连接的主要成分是连接蛋白。这个项目预计将描述一种新的缝隙连接在细胞表面组装的机制。该项目将确定连接蛋白在形成新的功能缝隙连接通道时可以回收和再利用的程度。它还将确定调节连接蛋白循环与降解的分子机制。综上所述，这将有助于更好地了解连接蛋白的周转和贩运。这些知识将有助于理解许多细胞现象，在这些现象中，细胞间的交流是至关重要的。实验收集的数据还将用于开发软件，该软件将从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隔间。该项目更广泛的影响有三个方面，即扩大对科学的参与，创造新的面向科学的艺术展览，以及提供研究培训。通过为匹兹堡大学的个人、匹兹堡周围广大农村地区的较小学院和少数族裔服务机构提供参与创新研究机会的机会，将加强对科学的参与，否则他们将无法获得这些机会。将为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。他们将在使用尖端成像和数学建模工具研究蛋白质动力学、循环和降解方面获得实践培训。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，学生将被指导创作描绘蛋白质动态和受调控的细胞行为的装置艺术。这些艺术展品将在工作坊、社区和当地美术馆展出。这些展品旨在鼓励在科学、技术、工程和数学(STEM)领域的职业发展。此外，他们还将向公众介绍和熟悉研究领域，突出活细胞的美丽和复杂性。缝隙连接的主要蛋白质亚基是连接蛋白，其中含量最丰富的是连接蛋白43。该项目将测试连接蛋白43的循环有助于形成新的缝隙连接通道的假设。具体地说，这项提议的第一个目标将确定多种细胞类型的内吞作用后连接蛋白43的动态变化。在第二个目标中，将检验这一假设，即连接蛋白的内吞作用和循环可以受到多种激酶和磷酸酶的调节。为了验证这些假说，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，并结合磷光体和药物激活剂。目标3将使用内吞作用、循环和降解的动态参数来生成一个微分速率方程模型，该模型将描述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，这最终是理解复杂的生理过程所必需的，从而有助于公共福利。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

Protocols for Generating Surfaces and Measuring 3D Organelle Morphology Using Amira.

• DOI: 10.3390/cells11010065
• 发表时间: 2021-12-27
• 期刊: Cells
• 影响因子: 6
• 作者: Garza-Lopez E;Vue Z;Katti P;Neikirk K;Biete M;Lam J;Beasley HK;Marshall AG;Rodman TA;Christensen TA;Salisbury JL;Vang L;Mungai M;AshShareef S;Murray SA;Shao J;Streeter J;Glancy B;Pereira RO;Abel ED;Hinton A Jr
• 通讯作者: Hinton A Jr

A Universal Approach to Analyzing Transmission Electron Microscopy with ImageJ.

• DOI: 10.3390/cells10092177
• 发表时间: 2021-08-24
• 期刊: Cells
• 影响因子: 6
• 作者: Lam J;Katti P;Biete M;Mungai M;AshShareef S;Neikirk K;Garza Lopez E;Vue Z;Christensen TA;Beasley HK;Rodman TA;Murray SA;Salisbury JL;Glancy B;Shao J;Pereira RO;Abel ED;Hinton A Jr
• 通讯作者: Hinton A Jr

Toxic stress and burnout: John Henryism and social dominance in the laboratory and STEM workforce.

有毒的压力和倦怠：约翰·亨利主义和实验室和STEM劳动力中的社会主导地位。

• DOI: 10.1093/femspd/ftab041
• 发表时间: 2021-09-11
• 期刊: Pathogens and disease
• 影响因子: 3.3
• 作者: Rolle T;Vue Z;Murray SA;Shareef SA;Shuler HD;Beasley HK;Marshall AG;Hinton A
• 通讯作者: Hinton A

Time management for STEMM students during the continuing pandemic.

• DOI: 10.1016/j.tibs.2021.12.010
• 发表时间: 2022-04
• 期刊: TRENDS IN BIOCHEMICAL SCIENCES
• 影响因子: 13.8
• 作者: Murray, Sandra A.;Davis, Jamaine;Shuler, Haysetta D.;Spencer, Elsie C.;Hinton, Antentor, Jr.
• 通讯作者: Hinton, Antentor, Jr.

Developing cultural humility in immunology and STEMM mentoring.

• DOI: 10.1016/j.it.2022.01.010
• 发表时间: 2022-04
• 期刊: TRENDS IN IMMUNOLOGY
• 影响因子: 16.8
• 作者: Murray, Sandra A.;Hinton, Antentor, Jr.;Spencer, Elsie C.
• 通讯作者: Spencer, Elsie C.