JNK regulation of desmosomes in development.

描述(申请人提供)：在发育过程中，表皮保护胚胎免受化学、生物和机械压力。这个器官在提供张力方面也是必不可少的，这是塑造器官所必需的，同时也允许形态发生所需的细胞形状变化。表皮的完整性是由包括桥粒在内的细胞间连接复合体所调节的。然而，人们对桥粒在胚胎发育过程中是如何调控的知之甚少。我的实验室已经发现了c-Jun NH(2)-末端激酶(JNK)信号在调节非洲爪哇蝌蚪表皮中桥粒蛋白方面的潜在新作用。JNK信号已被证明在胚胎发育过程中调节多种基因的转录或直接磷酸化细胞骨架蛋白。但是，这个MAPK家族成员从未被证明对桥粒功能有调节作用。我们发现，JNK信号的降低会导致符合人类情况的表型，这些表型是由桥粒功能缺陷引起的，如表皮脆性和起泡，以及心脏和颅面缺陷。此外，在JNK靶点的蛋白质组学筛查中，我们发现了两种Plakin：与哺乳动物的桥粒相关的外膜蛋白和周膜蛋白。这一建议旨在使用多种方法的组合来实现表皮中JNK信号功能的时间和空间丧失。接下来将描述整个胚胎和细胞水平上的影响。我们将使用反义技术、光激活和移植试验来实现JNK1功能的表皮特异性丧失。桥粒将通过电子显微镜和共聚焦显微镜进行分析。最后，我们的目标是通过定量聚合酶链式反应和免疫共沉淀来确定JNK是通过转录间接还是通过直接磷酸化来调节桥粒蛋白，表型蛋白和周型蛋白。我相信这是一项创新的赠款，它使用了完整胚胎的方法，并将发育信号和桥粒功能联系起来。

DESCRIPTION (provided by applicant): During development the epidermis protects the embryo from chemical, biological and mechanical stresses. This organ is also essential in providing tension, necessary for shaping organs while also allowing for cell shape changes necessary for morphogenesis. The integrity of the epidermis is mediated by intercellular junctional complexes, including desmosomes. However, relatively little is known about how desmosomes are regulated during embryonic development. My lab has uncovered a potentially new role for c-Jun NH(2)- terminal kinase (JNK) signaling in regulating desmosomal proteins in the epidermis of Xenopus laevis tadpoles. JNK signaling has been shown to regulate transcription of a variety of genes or to directly phosphorylate cytoskeletal proteins during embryogenesis. But, this MAP kinase family member has never been shown to regulate desmosomal function. We have found that decreased JNK signaling results phenotypes consistent with human conditions resulting from defective desmosomal function such as epidermal fragility and bubbling as well as heart and craniofacial defects. Further, in a proteomics screen for JNK targets we uncovered two plakins; epiplakin and periplakin that are associated with desmosomes in mammals. This proposal aims to use a combination of methods to perform temporal and spatial loss of function of JNK signaling in the epidermis. This will be followed by characterizing the effects in the whole embryo and at the cellular level. We will perform epidermal specific loss of JNK1 function using antisense technology, photoactivation and transplant assays. Desmosomes will be analyzed by electron and confocal microscopy. Finally we aim to determine whether JNK regulates the desmosomal proteins, epiplakin and periplakin, indirectly via transcription or by direct phosphorylation using quantitative PCR and co-immunoprecipitations. I believe this to be an innovative grant that uses whole embryo approaches and bridges developmental signaling and desmosomal function.