CX43 IN MITOSIS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 细胞周期通过维持恒定的体积和动态平衡来帮助调节细胞生长。缝隙连接(GJS)是这一过程中不可或缺的一部分，因为细胞部分地通过细胞间的通讯来帮助维持细胞内统一的代谢物和离子浓度。有丝分裂是细胞周期中不可或缺的一部分，在整个细胞中引起显著的形态和生化变化。当然，当组织培养细胞进入有丝分裂并经历细胞圆化和胞质分裂时，GJ结构形态和分布的一些最大变化发生在它们的GJ内化。在未受刺激(正常运输细胞)的NRK细胞中，从免疫沉淀细胞裂解物中分离的Cx43在Western blotts上显示三条带。它们包括一种非磷酸化形式(NP)和两种磷酸化形式(P1和P2)，它们主要在多个未鉴定的丝氨酸位点上磷酸化。GJ斑块主要含有P1型和P2型，而NP型主要分布在细胞内。使用免疫荧光，诺可达唑同步有丝分裂细胞显示Cx43完全在细胞内定位。在这些有丝分裂细胞中发现的Cx43是唯一被磷酸化的，并以p34cdc2/Cyclin B依赖的方式作为一个独特的P3物种迁移。大多数缝隙连接蛋白都含有多个磷酸化位点。这些磷酸化位点通常位于C-末端区域，尽管Cx56在细胞质环中还有一个额外的磷酸化位点。这些磷酸化位点对蛋白质的正常运输、组装和降解非常重要，并可能在信号转导中发挥作用，因为这些连接蛋白含有几个蛋白激酶共同的磷酸化序列。尽管如此，羧基末端可以被删除，GJS仍然可以组装，因此磷酸化位点在组装中的意义尚不清楚。用蛋白酶体、溶酶体和蛋白质合成抑制剂处理诺康唑阻断后释放的细胞表明，蛋白质降解在有丝分裂后P3-Cx43的消失中起作用。虽然在有丝分裂后GJS的重塑过程中会有大量的蛋白质合成，但来自活细胞成像的有趣证据表明，一些连接蛋白的循环也可能发生。在光学显微镜分辨率水平上，没有关于连接蛋白结构性质的形态信息，因为细胞内的定位表现为小的亮点，并且新旧蛋白质不能相互区分。这个项目的重点是确定有丝分裂期间是否发生循环，并使用四半胱氨酸技术来做到这一点。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The cell cycle helps to regulate cell growth by maintaining constant volumes and homeostasis. Gap junctions (GJs) are an integral part of this process because it is partially through intercellular communication that cells help to maintain uniform cytosolic metabolite and ion concentrations. Mitosis, an integral part of the cell cycle, causes significant morphological and biochemical changes throughout the entire cell. Certainly, some of the biggest changes in GJ structure morphology and distribution occur as tissue culture cells internalize their GJs as they go into mitosis and undergo cell rounding and cytokinesis. In unstimulated (normally trafficking cells) NRK cells, Cx43 isolated from immunoprecipitated cell lysates show three bands on Western blots. These include a nonphosphorylated form (NP) and two phosphorylated forms (P1 and P2) that are predominately phosphorylated on multiple, unidentified serine sites. GJ plaques contain predominantly the P1 and P2 forms while the NP form is localized intracellularly. Using immunofluorescence, nocodazole synchronized mitotic cells display an entirely intracellular localization of Cx43. The Cx43 found in these mitotic cells is uniquely phosphorylated and migrates as a distinct P3 species in a p34cdc2/cyclin B kinase-dependent manner. Most connexins contain sites for multiple sites for phosphorylation. These phosphorylation sites are typically found in the C-terminal region, although Cx56 has an additional phosphorylation site located in the cytoplasmic loop. These phosphorylation sites are important for proper protein trafficking, assembly and degradation and likely play a role in signal transduction as these connexins contain several protein kinase consensus phosphorylation sequence. Nonetheless, the carboxyl terminus can be deleted and GJs will still assemble so the significance of phosphorylation sites in assembly is not yet understood. Treatment of cells released from nocodazole block with proteosomal, lysosomal and protein synthesis inhibitors demonstrated that protein degradation plays a role in the disappearance of the P3-Cx43 after mitosis. While substantial protein synthesis occurs during reformation of GJs after mitosis, there is intriguing evidence from live cell imaging that some connexin recycling may also be occurring. At the light microscopy resolution level, there is no morphology information on the nature of connexin bearing structures since the intracellular localization appears as small bright spots and old versus new protein cannot be distinguished from each other. This project is focused on determining whether recycling occurs during mitosis and uses the tetracysteine technology to do so.