Investigation of the Regulation of Annular Gap Junction Processing, a Critical Modulator of Cell-Cell Communication

相邻的细胞之间需要相互沟通，以便正常运作，维持适当的组织结构，并对环境的变化做出适当的反应。一种通讯方法涉及通过聚集在相邻细胞表面的膜通道直接传递信息。这些通道簇被称为缝隙连接斑块，它们由称为连接蛋白的蛋白质组成。细胞表面缝隙连接斑块的去除是维持细胞间通讯的关键，并影响细胞的许多功能，包括增殖、细胞迁移和分化。该项目将阐明去除缝隙连接斑块所需的步骤，并确定潜在的分子机制。增加关于环状缝隙连接处理和降解的信息可以促进新技术的发展，以影响和控制细胞行为，包括增殖和激素反应。PI还将继续作为培训本科生、研究生和少数民族院校客座教授的资源。她用过去的NSF研究经费培训了45名本科生、8名研究生、3名博士后研究员、7名少数民族院校的客座教授、5名高中生和10名医科学生。多年来，参与这项研究的大多数学生都是非洲裔美国人，这是一个在科学界代表性严重不足的群体。她还将继续通过面向科学界的出版物、研讨会、讲座和工作坊进行更广泛的推广，并通过在当地艺术博物馆的艺术展示和讲座向公众进行独特的推广。这个项目将调查监管环形缝隙连接加工的机制。PI假设，通过磷酸化缝隙连接通道蛋白C端尾部的位点，连接蛋白43(Cx43)在降解所需的环状缝隙连接囊泡加工中发挥关键作用。这种加工包括环状缝隙连接分裂以及自体和多囊内体的形成。肾上腺皮质细胞群将被用来阐明激酶介导的Cx43磷酸化在环状缝隙连接处理和降解中的作用。该方法将操纵三种已知的与环状缝隙连接降解有关的蛋白激酶[蛋白激酶C(PKC)，Src，cAMP依赖的蛋白激酶(PKA)]来磷酸化Cx43。在目标I中，测量PKA、PKC和Src活性发生改变的细胞群体中环形缝隙连接囊泡的降解过程。三种互补的方法将被用来选择性地降低或增加蛋白激酶活性：1)用一组试剂进行化学处理；2)敲除(SiRNA)方案；以及3)感染复制缺陷的腺病毒载体，导致结构性激活的激酶。目的II测定Cx43磷酸化位点发生改变的细胞内环隙连接囊泡的降解过程。连接蛋白突变和截断技术将被用来修饰Cx43蛋白细胞内“尾部”的区域，该区域被认为是激酶结合和/或磷酸化的关键。在整个研究过程中，将用免疫细胞化学、量子点免疫电子显微镜、免疫印迹和时间推移图像分析来研究环状缝隙连接处理(分裂、自体和多囊内小体的形成以及与其他细胞器(溶酶体、内小体和质膜[可能将Cx43循环到质膜])的融合。针对靶点的磷酸特异性抗体已经可用，并将用于成像不同Cx43磷酸盐的时空分布和蛋白质含量。该实验室将继续作为培训本科生、研究生和少数族裔机构客座教授的资源。学生们将被分配一个特定的项目，并将被鼓励撰写并向科学界和普通公众展示他们的发现。国际和平协会将继续通过在国内和国际上发表出版物、研讨会、讲座和讲习班对科学界产生影响。此外，由PI创作并在当地美术馆展出的讲座和装置艺术展示将使PI能够向普通公众传授动态细胞事件。希望从这项研究中获得的信息和提供的培训将有助于促进科学，从而为公共福利做出贡献。

Neighboring cells need to communicate with one another in order to function properly, maintain proper organization of tissues, and make appropriate responses to changes in their environments. One method of communication involves the direct flow of information through membrane channels that are clustered on the surface of adjacent cells. These clusters of channels are called gap junction plaques and they are composed of proteins called connexin. The removal of gap junction plaques, from the cell surface, is critical to maintaining cell-to-cell communication and impacts numerous cellular functions, including proliferation, cell migration and differentiation. This project will elucidate the needed steps in gap junction plaque removal and determine the underlying molecular mechanisms. Increased information on annular gap junction processing and degradation could facilitate the development of new techniques to influence and control cell behavior, including proliferation and hormonal response. The PI will also continue to serve as a resource for the training of undergraduates, graduate students, and visiting professors from minority institutions. She used past NSF research funding to train forty-five undergraduates, eight graduate students, three postdoctoral fellows, seven visiting professors from minority institutions, five high school students, and ten medical students. Most of the students participating in this research over the years have been African-Americans, a severely under-represented group in science. She will also continue her broader outreach through publications, seminars, lectures and workshops for the scientific community and a unique outreach to the general public through art displays and lectures at a local art museum.This project will investigate the mechanisms regulating annular gap junction processing. The PI hypothesizes that protein kinases via phosphorylation of sites on the C-terminal tail of the gap junction channel protein, connexin 43 (Cx43), play a pivotal role in annular gap junction vesicle processing needed for degradation. Such processing involves annular gap junction fissions as well as the formation of autophagasomes and multivesicular endosomes. Adrenal cortical cell populations will be used to elucidate the role of kinase-mediated Cx43 phosphorylation in annular gap junction processing and degradation. The approach will be to manipulate three kinases [protein kinase C (PKC), Src, cAMP-dependent protein kinase (PKA)] known to phosphorylate Cx43 and thought to be involved in annular gap junction degradation. In Objective I, To Measure the Annular Gap Junction Vesicle Degradative Process in Cell Populations in Which PKA, PKC, and Src Activities Have Been Altered. Three complementary methodologies will be used to selectively decrease or increase protein kinase activity: 1) chemical treatments with a panel of agents; 2) knockdown (siRNA) protocols; and 3) infection with replication-defective adenovirus vectors which result in constitutively activated kinases. In Objective II To Measure Annular Gap Junction Vesicle Degradative Process in Cells in Which the Cx43 Phosphorylation Sites Have Been Altered. Connexin mutation and truncation techniques will be used to modify the region of the intracellular "tail" of the Cx43 protein thought to be critical for kinase binding and/or phosphorylation. Throughout this study, annular gap junction processing (fission, formation of autophagasomes and multivesicular endosomes and fusion with other organelles (lysosomes, endosomes and plasma membranes [possible recycling of Cx43 to the plasma membrane]) will be investigated with immunocytochemical, quantum dot immuno-electron microscopy, western blot, and time lapse image analysis. Phospho-specific antibodies against the targeted sites are available and will be used to image the spacial and temporal distribution as well as protein amounts of the differing Cx43phosphoforms.The laboratory will continue to serve as a resource for the training of undergraduates, graduate students, and visiting professors from minority institutions. Students will be given a specific project and they will be encouraged to write and present their findings, both to the scientific community and to the general public. The PI will continue to make an impact on the scientific community through publications, seminars, lectures and workshops presented both nationally and internationally. In addition, lectures and installation art displays created, by the PI and displayed at a local art museum, will enable the PI to teach the general public about dynamic cellular events. It is hoped that information gained from this study and the training provided will serve to advance science and thus contribute to public welfare.