Connecting TOR signalling with proliferation through the RETINOBLASTOMA RELATED complex in Arabidopsis root meristem

考虑到农业生产力从根本上取决于营养物质、碳和能量如何支撑植物生长，人们对一些潜在的生物过程知之甚少。依赖于蛋白质翻译的细胞生长限制了细胞增殖。我们实验室的新数据表明，由雷帕霉素（TOR）和S6激酶（S6 K）靶点直接发出的生长信号，以及通过EBP 1协调的核糖体生物发生和翻译控制，与以视网膜母细胞瘤相关（RBR）蛋白和下游转录因子为中心的细胞周期控制途径有关。进一步了解的调节途径，蛋白质翻译和细胞增殖，以调节器官生长，需要系统的方法，在特定的细胞类型中，这些途径是可操作的，即增殖分生组织细胞。在这个项目中，我们专注于拟南芥根，其中的分生组织和延伸结构域是明确的，并访问显微镜观察，分析突变体和收集细胞特异性的定量数据的计算modeling.In任务1，我们将测量随着时间的推移，根生长速度的函数在幼苗的根生长的细胞和分子标记。生长速率将通过调节蔗糖浓度、光照强度来操纵。我们将从DNA合成的时程EdU标记开始，以确定细胞周期长度的变化。然后，我们将使用荧光标记的细胞核和细胞膜以及使用共聚焦显微镜的S-G2和M期的双色细胞周期标记物来建立细胞周期进程的活根成像。我们将使用翻译报告基因来确定蛋白质翻译的动态变化。首先，我们将关注拟南芥野生型根分生组织如何响应蔗糖可用性，然后我们将在TOR、RAPTOR、S6 K1和S6 K2、RPS 6、eIF 3 h、EBP 1的突变/沉默系中进行生长定量，并使用TOR的化学抑制。我们的目标是证实我们在固定准备或通过切割实时成像收集的数据-由伦敦帝国学院塞纳博士指导的植物形态发生实验室提供的边缘光片显微镜。该仪器由Sena博士及其同事专门开发，用于拟南芥根活体荧光成像，具有高空间（微米）和时间（分钟）分辨率，可进行长时间（天）观察。项目的这一部分将侧重于任务1中确定的一些选定的关键参数和条件。任务1和任务2中的实验结果将是关于蛋白质翻译、细胞周期参数、细胞和分生组织大小的动态、定量、细胞分辨率数据，在任务3中，我们将建立在我们的专业知识，确定了TOR信号通路的调节靶点，提供调节输入控制细胞周期转换点，从而控制细胞周期参数、分生组织大小和根生长速率。在任务3中，我们将使用转录报告子（启动子-报告子）、翻译报告子（启动子-UTR-报告子）和功能性翻译基因融合体（启动子-基因-报告子）收集网络内调控分子的细胞特异性动态数据。我们将图像的幼苗根共聚焦显微镜，并在选定的记者长时间观察，我们也将使用光片显微镜。此外，我们将确定磷酸化状态的S6 K，RBR特别是在根分生组织和延伸区使用域特异性启动子表达GFP标记的RBR S6 K1。我们还将定量测量RBR-E2 FA和RBR-E2 FB相互作用的免疫共沉淀在这些领域。这些定量数据将有助于对监管网络进行建模。

Considering that agricultural productivity is fundamentally dependent on how nutrients, carbon and energy underpin plant growth, remarkably little is known about some of the underlying biological processes. Cell growth, which relies on protein translation, constrains cell proliferation. Emerging data in our laboratory suggest that growth signalling by the TARGET OF RAPAMYCIN (TOR) and S6 KINASE (S6K) directly, and through the ribosome biogenesis and translational control orchestrated by EBP1 are connected with the cell cycle control pathways centred on the RETINOBLASTOMA-RELATED (RBR) protein and the downstream transcription factors. Further understanding of the regulatory pathways that link protein translation and cell proliferation to regulate organ growth requires systems approaches in the specific cell types in which these pathways are operational, namely proliferating meristematic cells. In this project, we focus on the Arabidopsis root, where the meristematic and elongation domains are well defined and accessible for microscopic observations to analyze mutants and to collect cell specific quantitative data for computational modelling.In task 1 we shall measure cellular and molecular markers of root growth in seedlings over time as a function of root growth rate. The growth rate will be manipulated by adjusting the sucrose concentration, light intensity. We will start with time course EdU labelling of DNA synthesis to determine the change in cell cycle length. Then we shall set up live root imaging of cell cycle progression using fluorescently labelled nuclei and cell membrane and dual colour cell cycle markers for S-G2 and M phases using confocal microscopy. We shall use translational reporters to determine dynamic changes in protein translation. First we will focus on how Arabidopsis wild-type root meristems respond to sucrose availability and then we shall perform growth quantitation in mutant/silenced lines of TOR, RAPTOR, S6K1 and S6K2, RPS6, eIF3h, EBP1, and use chemical inhibition of TOR.In task 2, we aim to corroborate the data we collect on fixed preparation or by live imaging with the cutting-edge light sheet microscope available in the Laboratory of Plant Morphogenesis directed by Dr Sena at Imperial College London. This instrument has been specifically developed by Dr Sena and colleagues for Arabidopsis root live fluorescence imaging, at high spatial (microns) and temporal (minutes) resolutions and for very long (days) observations. This part of the project will focus on some selected critical parameters and conditions determined in task 1. The outcome of the experiments in tasks 1 and 2 will be dynamic, quantitative, data with cellular resolution on protein translation, cell cycle parameters, cell and meristem sizes, and growth rates under a range of conditions and TOR pathway activities that either stimulate or repress cell proliferation.In task 3 we shall build on our expertise that identified regulatory targets of the TOR signalling pathway that provide regulatory inputs that controls cell cycle transition points and consequently the cell cycle parameters, meristem size and root growth rate. Within task 3 we will collect cell specific dynamic data on the regulatory molecules within the network using transcriptional reporters (promoter-reporter), translational reporters (promoter-UTR-reporter) and functional translational gene fusions (promoter-gene-reporter). We will image the seedling root by confocal microscopy and, for long observations on selected reporters we will also use light sheet microscopy. Additionally, we will determine the phosphorylation state of S6K, RBR specifically in the root meristem and in the elongation zone using domain-specific promoters to express GFP-tagged RBR S6K1. We shall also quantitatively measure RBR-E2FA and RBR-E2FB interactions by co-immunoprecipitation in these domains. These quantitative data will allow modelling the regulatory network.