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

通过拟南芥根分生组织中的视网膜母细胞瘤相关复合体将 TOR 信号传导与增殖联系起来

• 批准号: 1813952
• 金额: --
• 依托单位: Royal Holloway University of London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1813952
• 关键词: Connecting; TOR; signalling; proliferation; through

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.

考虑到农业生产力从根本上取决于养分、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。细胞生长依赖于蛋白质翻译，限制了细胞增殖。我们实验室的新数据表明，雷帕霉素靶标 (TOR) 和 S6 激酶 (S6K) 直接产生的生长信号传导，以及通过 EBP1 精心策划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相关。进一步了解连接蛋白质翻译和细胞增殖以调节器官生长的调节途径，需要针对这些途径发挥作用的特定细胞类型（即增殖分生细胞）的系统方法。在这个项目中，我们重点关注拟南芥根，其中分生组织和伸长域被明确定义，并且可以进行微观观察，以分析突变体并收集细胞特定的定量数据以进行计算建模。在任务 1 中，我们将测量幼苗中根生长随时间变化的细胞和分子标记，作为根生长速率的函数。通过调节蔗糖浓度、光照强度来控制生长速率。我们将从 DNA 合成的时间进程 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 期双色细胞周期标记物建立细胞周期进展的活根成像。我们将使用翻译记者来确定蛋白质翻译的动态变化。首先，我们将重点关注拟南芥野生型根分生组织如何响应蔗糖可用性，然后我们将对 TOR、RAPTOR、S6K1 和 S6K2、RPS6、eIF3h、EBP1 的突变体/沉默系进行生长定量，并使用 TOR 的化学抑制。在任务 2 中，我们的目标是证实我们在固定制备或通过使用现有的尖端光片显微镜实时成像收集的数据。 伦敦帝国理工学院植物形态发生实验室由 Sena 博士领导。该仪器是由 Sena 博士及其同事专门开发的，用于拟南芥根部实时荧光成像，具有高空间（微米）和时间（分钟）分辨率，并可进行很长时间（数天）的观察。该项目的这一部分将重点关注任务 1 中确定的一些选定的关键参数和条件。任务 1 和 2 中的实验结果将是动态、定量的数据，具有细胞分辨率，包括蛋白质翻译、细胞周期参数、细胞和分生组织大小、一系列条件下的生长率以及刺激或抑制细胞增殖的 TOR 通路活动。在任务 3 中，我们将利用我们的专业知识来确定 TOR 信号传导的调控目标 途径提供控制细胞周期转变点的调节输入，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基因（启动子-报告基因）、翻译报告基因（启动子-UTR-报告基因）和功能翻译基因融合（启动子-基因-报告基因）收集网络内调节分子的细胞特异性动态数据。我们将通过共焦显微镜对幼苗根部进行成像，并且为了对选定的记者进行长期观察，我们还将使用光片显微镜。此外，我们将使用域特异性启动子表达 GFP 标记的 RBR S6K1，确定根分生组织和伸长区中 S6K、RBR 的磷酸化状态。我们还将通过免疫共沉淀在这些域中定量测量 RBR-E2FA 和 RBR-E2FB 相互作用。这些定量数据将允许对监管网络进行建模。

项目成果

Arabidopsis RETINOBLASTOMA RELATED directly regulates DNA damage responses through functions beyond cell cycle control.

• DOI: 10.15252/embj.201694561
• 发表时间: 2017-05-02
• 期刊: The EMBO journal
• 作者: Horvath BM;Kourova H;Nagy S;Nemeth E;Magyar Z;Papdi C;Ahmad Z;Sanchez-Perez GF;Perilli S;Blilou I;Pettkó-Szandtner A;Darula Z;Meszaros T;Binarova P;Bogre L;Scheres B
• 通讯作者: Scheres B