Gap Junction Plaque Internalization

细胞通过被称为间隙连接通道的膜上的孔相互交流。分子通过间隙连接通道的运动与人体大多数细胞的发育、分化和功能有关。通道的组装和聚集形成功能性的间隙连接斑块以及这些间隙连接斑块从细胞膜上的移除被认为是细胞行为的关键。间隙连接斑块的清除是通过一个独特的内化过程发生的，在这个过程中，两个相对的膜被合并到两个接触细胞中的一个的细胞质中，形成环形的间隙连接小泡。有强有力的证据表明，网格蛋白（一种已知参与膜蛋白内吞作用的蛋白质）和蛋白激酶参与了这一内化过程。在本研究中，假设是蛋白激酶介导的间隙连接通道蛋白（Connexin 43）磷酸化控制网格蛋白向间隙连接斑块募集，从而调节间隙连接斑块内化。为了验证这一假设，形态学方法（免疫细胞化学、活细胞成像、电子显微镜）和分子改变（截断和突变来修饰关键的激酶结合和/或间隙连接蛋白的磷酸化位点）将在肾上腺、子宫和卵巢细胞培养中使用。此外，蛋白激酶活性将通过化学处理、低敲（siRNA）方案、微发色团辅助snap标签蛋白的激光失活和复制缺陷腺病毒载体感染而改变。从这项研究中得到的信息将增加我们对间隙连接蛋白运输以及一般膜运输的理解。此外，阐明间隙连接内化的机制将影响我们针对间隙连接进行治疗以控制和/或纠正细胞行为的能力。更广泛的影响该实验室将继续为培养高中生、本科生和研究生以及少数民族院校的客座教授提供资源。受训者将参与该项目的各个方面，并鼓励他们在研究实验室中撰写并向科学界和公众展示他们的发现。除了获得细胞和分子生物学工具的指导和经验外，学生还将接受公开演讲，拨款写作，手稿准备，实验室操作和职业管理方面的培训。通过国内和国际的出版物、研讨会、讲座和讲习班，该研究小组有望对科学界产生影响。新颖的方法，如灌注艺术，用于演示细胞生物学过程，并用于吸引和教育公众。将继续发展和实施。

Cells communicate with one another through pores in the membrane called gap junction channels. Movement of molecules through the gap junction channels has been implicated in the development, differentiation and function of most cells of the body. The assembly and clustering of channels into functional gap junction plaques as well as the removal of these gap junction plaques from the cell membrane are thought to be critical to cell behavior. Gap junction plaque removal occurs by a unique internalization process in which the two opposing membranes are incorporated into the cytoplasm of one of two contacting cells, to form annular gap junction vesicles. There is strong evidence that clathrin (a protein known to be involved in endocytosis of membrane protein) and protein kinases are involved in this internalization process. In this research study, the hypothesis is that protein kinase-mediated phosphorylation of the gap junction channel protein (Connexin 43) control the recruitment of clathrin to the gap junction plaque and thus regulate gap junction plaque internalization. To test this hypothesis a combination of morphological methodologies (immunocytochemistry, live cell imaging, electron microscopy) and molecular alterations (truncations and mutations to modify critical kinase binding and/or phosphorylation sites on the gap junction protein) will be used in adrenal, uterine and ovarian cell cultures. In addition, protein kinase activity will be altered by chemical treatments, knockdown (siRNA) protocols, micro-chromophore-assisted laser inactivation of SNAP-tag proteins and infection with replication-defective adenovirus vectors. Information resulting from this study will increase our understanding of gap junction protein trafficking as well as of membrane trafficking in general. Furthermore, elucidating the mechanism of gap junction internalization will impact our capacity for targeting gap junctions for treatments to control and/or correct cell behavior.Broader ImpactThe laboratory will continue to serve as a resource for the training of high school, undergraduate and graduate students, as well as visiting professors from minority institutions. Trainees will be involved in all aspects of this project and they will be encouraged to write and present their findings in the research laboratory, both to the scientific community and to the general public. In addition to obtaining instructions and experience with the tools of cell and molecular biology, students will receive training in public speaking, grant writing, manuscript preparation, laboratory operations, and career management. Through publications, seminars, lectures and workshops, presented both nationally and internationally, the research team is expected to make an impact on the scientific community. Novel methods, such as instillation art, for demonstrating processes in cell biology and for engaging and educating the general public.will continue to be developed and implemented.