Mechanosensitive remodeling of adherens junctions during snail-driven EMT

摘要/项目摘要 粘附连接的主动重塑不仅对保持组织完整性至关重要，而且对驱动细胞粘附也至关重要。 许多发育事件的进展，如上皮-间充质转化（EMT）。 细胞生物学与机械力界面研究的最新进展， 揭示，除了充分研究的生化信号，机械张力起着关键作用。 但在调节粘附连接中的作用尚未得到重视。候选人的博士后研究 表明在果蝇原肠胚形成过程中，中胚层原基中的粘附连接是 重新定位和加强响应顶端局部肌球蛋白收缩。如此紧张- 粘附连接的依赖性重塑保护连接免受Snail驱动的连接解体 并且似乎在发育时间上EMT仅发生在形态发生完成之后， 活动这建立了一个模型来研究机械敏感性调节的粘附连接在一个 完整的发育系统。该项目的目标是阐明张力依赖的机制， EMT背景下的粘附连接重塑。在第一个目标中， 在重塑过程中，将使用 光转换荧光探针和体内连接张力将在活胚胎中使用 FLIM（荧光寿命成像显微镜）基于FRET张力传感器， 最近制造并测试的在第二个目标中，一个小而全面的直接蜗牛目标池 从最近的一项全基因组研究中鉴定出的基因将被测试它们在依赖蜗牛的 接头拆卸。将在独立阶段进一步表征鉴定的基因。的 最后一个目的是测试张力依赖性连接重塑的原理及其对EMT的影响， 脊椎动物细胞系的研究。稳定表达活性的细胞株 将在指导阶段制作成像标记。结动力学和 将在独立阶段检查EMT期间的机械敏感性连接重塑。结果 从这个项目将促进我们对粘附连接的调节的理解， 肌球蛋白产生的张力，揭示了Snail在分解连接中的新功能， 转录后水平。

Abstract/Project Summary Active remodeling of adherens junctions is crucial not only for keeping tissue integrity but also for driving the progression of many developmental events such as epithelial-mesenchymal-transition (EMT). Recent development in the research at the interface between cell biology and mechanical force has revealed that, in addition to the well studied biochemical signals, the mechanical tension plays an critical yet unappreciated role in the regulation of adherens junctions. The candidate's postdoc study has demonstrated that during Drosophila gastrulation adherens junctions in mesodermal primordium are repositioned and strengthened in response to apically localized myosin contraction. Such tension- dependent remodeling of adherens junctions protects junctions from Snail-driven junction disassembly and appear to developmentally time the EMT to occur only after the completion of the morphogenetic event. This establishes a model to study the mechanosensitive regulation of adherens junctions in an intact developmental system. The goal of the project is to elucidate the mechanism of tension-dependent adherens junction remodeling in the context of EMT. In the first aim, the dynamics of junctional components within individual junction clusters during the remodeling will be examined using photoconvertible fluorescent probes and in vivo junctional tension will be measured in live embryos using a FLIM (Fluorescence Lifetime Imaging Microscopy)-based FRET tension sensor that have been recently made and tested. In the second aim, a small yet comprehensive pool of direct Snail target genes identified from a recent genome-wide study will be tested for their roles in Snail-dependent junction disassembly. The identified genes will be further characterized in the independent phase. The last aim is to test the principle of tension-dependent junction remodeling and its impact on EMT in vertebrate cell lines in collaboration with McClatchey's lab. The cell lines with stably expressed live imaging markers will be made during the mentored phase. Junction dynamics and the role of mechanosensitive junction remodeling during EMT will be examined in the independent phase. Result from this project will advance our understanding on regulation of adherens junctions in response to myosin-generated tension and shed light on the novel function of Snail on disassembling junctions at post-transcriptional level during EMT.