Analysis of keratin dynamics in the early mouse embryo: regulatory mechanisms and functional consequences

上皮角蛋白骨架通过与桥粒、肌动蛋白骨架和相关蛋白的相互作用，对细胞粘附和细胞类型特异性功能至关重要。角蛋白的结构和相互作用受Src、PKCalpha和MAPK家族成员响应细胞内和细胞外信号的时空调控。尽管角蛋白在胚胎发育、伤口愈合和肿瘤进展中的重要性得到了充分的证明，但迄今为止，角蛋白网络的组装和动力学及其调控仅在体外进行了研究。尽管形态学和遗传学证据支持角蛋白、桥粒和肌动蛋白之间的相互依存关系，但尚未在体内研究角蛋白网络新生形成的机制及其在分化、粘附和侵袭过程中的调节和意义。我们的目标是揭示角蛋白丝网络，桥粒和肌动蛋白组织在小鼠胚胎生理背景下的相互作用。该建议是基于我们实验室对角质形成细胞中角蛋白网络调节的广泛合作工作。在此，我们希望利用自荧光角蛋白8 K8-YFP作为一种新的体内检测工具，分析植入前和植入过程中活体小鼠胚胎滋养外胚层角蛋白网络的形成、动态、功能和调控。在我们最近建立的K8-YFP敲入小鼠的早期胚胎（桑葚胚期到囊胚期）和胚胎源性滋养细胞系中，我们将追求三个主要目标：1。活体小鼠胚胎新生角蛋白网络形成的分析滋养细胞中角蛋白-桥粒支架在植入和侵袭过程中的动态组织和功能的研究利用药理学和遗传学方法在培养的滋养细胞和发育中的囊胚中调控角蛋白网络的生物发生、组织和相互作用的分子机制分析我们期望我们的项目通过一个可访问的体内模型系统，为体内角蛋白细胞骨架的时空组织和调节提供新的机制见解。从长远来看，我们的方法为解决伤口愈合和肿瘤转移过程中角蛋白的动态调节提供了基础，在这种情况下，理解角蛋白-桥粒重组的原理可能提供新的治疗靶点。

The epithelial keratin cytoskeleton is crucial for cell adhesion and cell type-specific functions through interactions with desmosomes, the actin cytoskeleton and associated proteins. Architecture and interactions of keratins are spatiotemporally regulated by Src, PKCalpha and MAPK family members in response to intra- and extracellular signals. Despite the well-documented importance of keratins in embryo development, wound healing and tumor progression, assembly and dynamics of the keratin network and its regulation have so far been studied only in vitro. Although morphological and genetic evidence supports interdependence between keratins, desmosomes and actin, the mechanism underlying de novo keratin network formation, its regulation and significance during differentiation, adhesion and invasion have not been investigated in vivo. Our goal is to unravel interactions between the keratin filament network, desmosomes and actin organization in the physiological context of mouse embryos. The proposal is based on extensive collaborative work from our laboratories on keratin network regulation in keratinocytes. Here, we want to analyze keratin network formation, dynamics, function and regulation in the trophectoderm of live mouse embryos before and during implantation using auto-fluorescent keratin 8 K8-YFP as a novel in vivo detection tool.In early embryos (morula to blastocyst stages) and embryo-derived trophoblast cell lines from K8-YFP knock-in mice, that we recently established, we will pursue three major objectives: 1. Analysis of de novo keratin network formation in live murine embryos2. Examination of the dynamic organization and function of the keratin-desmosome scaffold in the trophoblast during implantation and invasion 3. Analysis of molecular mechanisms regulating keratin network biogenesis, organization and interactions using pharmacological and genetic approaches in cultured trophoblast cells and developing blastocystsWe expect that our project provides novel mechanistic insights into the spatiotemporal organization and regulation of the keratin cytoskeleton in vivo, using an accessible in vivo model system. In the long run, our approach provides the ground to address dynamic keratin regulation during wound healing and during tumor metastasis, conditions in which understanding principles of keratin-desmosome reorganization likely provide novel therapeutic targets.