Analysis and reconstitution of regulatory networks governing epidermal differentiation and polar root hair growth

顶端聚焦生长是建立细胞极性的重要机制，也是神经元轴突、真菌菌丝和植物根毛生长的基本过程。根毛是特殊的表皮细胞的圆柱形细胞突起。根毛的极尖生长需要细胞骨架动力学、囊泡运输和离子通量的紧密协调和调节。在根毛中，高尖端的钙离子梯度已被证明是极地生长所必需的。钙离子的振荡增加叠加在钙离子梯度上，并可能协调针尖的振荡生长和活性氧簇(ROS)浓度和pH值的振荡波动。该项目的长期目标是了解极性RH尖端生长的分子组成和机制。此外，我们打算重建这样一个振荡网络。我们的初步数据和已发表的研究结果表明，钙离子激活的CBL/CIPK复合体和CDPKs同时激活NADPH氧化酶。此外，越来越多的证据还表明，钙离子非依赖的磷酸化是NADPH氧化酶激活的一种替代方式，以及特定的磷酸酶在抵消这种激活中的作用。在这个项目中，我们想要阐明信号网络的不同组成部分到底是如何发挥作用的，它们是如何动态相互连接的，以及钙参数的定量变化如何控制根毛中的信息处理。考虑到我们在过去几年中取得的新发现和结果，我们现在特别想重点关注以下研究目标：(I)探索RHS中以尖端为中心的钙梯度和钙振荡的产生机制和成分。(Ii)阐明钙离子依赖的磷酸化在调节RbohC活性中的作用。(Iii)研究钙非依赖性磷酸化对RbohC调控的潜在贡献。(4)探讨目前难以捉摸的导致RHS中RbohC失活所需的组成部分和机制。通过结合从项目领域(I)-(Iv)获得的见解，我们随后打算(V)表征这些激酶和磷酸酶在调节RbohC中的复杂相互作用，以及(Vi)启动调控网络的重建，该网络是在HEK293T细胞中异源形成钙振荡和ROS信号的基础。作为对这些方法的补充，我们打算进一步推进我们对RHS中第二信使动力学的研究，并继续寻找有助于这些过程的新成分。总而言之，我们的方法应该揭示这些信号组件在RH生长过程中复杂的相互作用的详细和基本的见解。

Tip-focused growth represents an important mechanism to establish cellular polarity and is a fundamental process that underlies the growth of neuronal axon, fungal hyphae and plant root hairs. Root hairs are cylindrical shaped cellular protuberances of specialized epidermal cells. Polar tip growth of root hairs requires tight coordination and regulation of cytoskeleton dynamics, vesicle transport and ion fluxes. In root hairs a high-tip Ca2+ gradient has been shown to be essential for polar growth. Oscillatory Ca2+ increases are superimposed on this Ca2+ gradient and are likely to coordinate oscillatory tip growth and oscillatory fluctuations of reactive oxygen species (ROS) concentrations and pH values. The long-term goal of this project is to understand the molecular components and mechanisms underlying polar RH tip growth. Moreover, we intend to reconstitute such an oscillating network. Our preliminary data as well as published findings indicate that Ca2+ activated CBL/CIPK complexes and CDPKs simultaneously activate NADPH oxidases. Moreover, accumulating evidence also suggests Ca2+ independent phosphorylation as an alternative means of NADPH oxidase activation and a role of specific phosphatases in counteracting such activation. In this project we want to elucidate how exactly the different components of signaling network function and how they are dynamically interconnected and how quantitative changes in Ca2+ parameters govern information processing in root hairs. Considering our new findings and results obtained during the last years, we now specifically want to focus on the following research aims: (i) Explore the mechanisms and components contributing to the generation of the tip-focused Ca2+ gradient and Ca2+ oscillations in RHs. (ii) Elucidate the role of Ca2+ dependent phosphorylation in modulating RbohC activity. (iii) Investigate a potential contribution of Ca2+-independent phosphorylation to the regulation of RbohC. (iv) Explore the currently elusive components and mechanisms that bring about the required deactivation of RbohC in RHs. By combining the insights gained from project areas (i) – (iv), we subsequently intend to (v) characterize the complex interplay of these kinases and phosphatases in regulating RbohC and (vi) initiate the reconstitution of a regulatory network underlying the formation of Ca2+ oscillations and ROS signals heterologously in HEK293T cells. Complementary to these approaches, we intend to further advance our investigations of second messenger dynamics in RHs and continue to identify novel components that contribute to these processes. Collectively, our approaches should reveal detailed and fundamental insights of the complex interplay of these signaling components in growing RH.