A New Paradigm for Cell Size Control by the RB Tumor Suppressor Pathway

描述（由申请人提供）：大小控制是细胞的基本特性，涉及生长和分裂的协调调节，这一过程在人类疾病（如癌症）中经常功能失调。这项研究的长期目标是建立控制尺寸的机械原理。我们已经开发了一个独特的信息模型的细胞大小调节，单细胞藻类衣藻莱茵。利用衣藻，我们已经确定了大小检查点途径的关键组分，其行为表明了细胞大小与细胞周期激活耦合的机制。该检查点的基因鉴定成分每个都有人类对应的成分，包括视网膜母细胞瘤（RB）肿瘤抑制途径的蛋白质，磷酸化RB的细胞周期蛋白依赖性激酶CDKG1和RNA结合蛋白TNY1。我们假设CDKG1激酶的活性受到TNY1的控制，作为细胞大小与细胞周期进入相耦合的一种手段，并且这两种活性的平衡通过打开和关闭RB通路来控制细胞周期。本提案的发现将提供关于细胞生长和分裂控制机制的基础知识，这与理解人类的类似过程有关。本建议的具体目标如下：目标1。研究CDKG1作为大小蛋白的作用：我们将i)确定CDKG1的丰度和活性如何在细胞周期中受到控制；ii)鉴定和表征CDKG1在其底物RB相关蛋白MAT3上的磷酸化位点；iii)利用条件等位基因确定细胞周期中对CDKG1的时间需求。目标2。确定TNY1抑制细胞周期的机制：每个细胞周期产生一次TNY1，然后随着细胞在G1期间的生长而被稀释。我们将测试TNY1作为细胞大小的阈值调节剂的作用，该调节剂反对CDKG1的活性。我们将i)确定TNY1是否通过结合CDKG1的mRNA调控CDKG1；ii)试验TNY1的剂量效应；iii)鉴定内源性TNY1 RNA靶点；iv)确定将TNY1靶向mRNA是否会使编码蛋白的表达受到TNY1和/或细胞周期的控制。目标3。通过抑制筛选确定新的大小途径基因：tny1零突变体和CDKG1过表达（CDKG1- oe）系都具有小细胞表型，提供了一个敏感的遗传背景，以确定大小控制的相互作用成分。i)我们将在正向筛选中使用这些细胞系来寻找其小细胞表型的抑制因子。据预测，这些抑制因子编码目标蛋白或相互作用蛋白。ii)我们将使用二次筛选来排序大小通路中的突变体，并确定候选突变体进行进一步的研究和克隆。

DESCRIPTION (provided by applicant): Size control is a fundamental property of cells that involves coordinated regulation of growth and division, processes that are frequently dysfunctional in human diseases such as cancer. The long-term goal of this research is to establish the mechanistic principles that govern size control. We have exploited a uniquely informative model for cell-size regulation, the unicellular alga Chlamydomonas reinhardtii. Using Chlamydomonas we have identified key components of a size checkpoint pathway whose behavior suggests a mechanism for coupling cell size to cell cycle activation. The genetically identified components of this checkpoint each have human counterparts and include proteins of the retinoblastoma (RB) tumor suppressor pathway, a cyclin dependent kinase, CDKG1, that phosphorylates RB, and an RNA binding protein, TNY1. We hypothesize that the activity of the kinase, CDKG1, is held in check by TNY1 as a means of coupling cell size to cell cycle entry, and that the balance of these two activities controls the cell cycle by switching on and off the RB pathway. The findings from this Proposal will provide fundamental knowledge regarding mechanisms of cell growth and division control that are relevant for understanding similar processes in humans. The Specific Aims of this Proposal are as follows: Aim 1. Investigate the role of CDKG1 as sizer protein: We will i) determine how CDKG1 abundance and activity are controlled during the cell cycle; ii) identify and characterize CDKG1 phosphorylation sites on its substrate, the RB related protein MAT3; iii) determine the temporal requirement for CDKG1 in the cell cycle using conditional alleles. Aim 2. Determine the mechanism by which TNY1 represses the cell cycle: TNY1 is produced once per cell cycle and then becomes diluted as cells grow during G1. We will test the role of TNY1 as a threshold regulator of cell size that opposes the activity of CDKG1. We will i) establish whether TNY1 regulates CDKG1 through binding its mRNA; ii) test dosage effects of TNY1; iii) identify endogenous TNY1 RNA targets; and iv) establish whether targeting TNY1 to a mRNA brings expression of the encoded protein under control of TNY1 and/or the cell cycle. Aim 3. Identify new size pathway genes through suppressor screens: tny1 null mutants and CDKG1 over- expressing (CDKG1-OE) lines both have small-cell phenotypes that provide a sensitized genetic background in which to identify interacting components of size control. i) We will use these lines in forward screens to find suppressors of their small-cell phenotypes. These suppressors are predicted to encode either targets or interacting proteins. ii) We will use secondary screens to order the mutants in the size pathway and to identify candidates for further investigation and cloning.