Determining how cell growth triggers cell division in epidermal stem cells

项目总结 这项提议旨在确定细胞生长如何触发细胞分裂，这是细胞中的一个基本问题 和发育生物学。它的理解也将极大地影响我们对癌症的知识，在那里 流程不规范。很早就知道，细胞生长在G1期触发人类细胞分裂/S 在复制DNA之前的过渡。但是，尽管许多将细胞生长与细胞分裂联系起来的关键调控蛋白 哺乳动物细胞用来控制其大小的分子机制仍然很差。 理解并一直完全基于对培养中细胞生长的研究。我的实验室最近做了一个 在理解增长如何引发分裂方面取得了突破性进展。与预期的增长将 增加Cyclin D-CDK4，6的活性，我们发现相反，细胞生长稀释了细胞周期抑制物Rb来触发 培养细胞的分裂。我们对细胞培养中Rb稀释机制的发现提出了三个关键问题 这项研究的重点是：1.调节Rb浓度动态的分子机制是什么 控制单元格大小？2.基于RB的单元格大小控制的功能是什么？3.RB稀释或其他单元格大小 控制机制将细胞生长与体内的细胞分裂联系起来。我们已经开始解决第一个问题，我们的 初步数据表明，调节Rb浓度大小依赖的机制是平移的。 为了进一步确定这种分子机制是如何工作的，我们将采取一种方法，使用记者来识别 DNA序列元件负责，然后相应的蛋白质调节其功能。致信地址 第二个问题，我们将采用基于质谱学的方法来测量蛋白质浓度 随着整个蛋白质组的细胞大小而变化。初步数据表明，与衰老有关的蛋白质 在大细胞中，表型浓度增加。这表明细胞大小可能是衰老的原因。 而细胞大小控制功能可以避免这种有害的结果。要解决最后一个最终要测试的问题 对于Rb稀释和替代模型，我们将进行一系列体内实验。这一点很重要，因为 最近在细胞培养方面的研究报告了关于动物细胞如何控制其大小的相互矛盾的结果。至 为了确定动物细胞生长如何在体内触发细胞分裂，我们建议研究小鼠的表皮 因为它有大量的增殖干细胞，它们的分裂动态可以用活的- 细胞成像。我们将测量RB家族在一系列小鼠系中角质形成细胞大小的变化 的基因在小鼠表皮中有条件地删除或过度表达。这将考验我们的中央 假设Rb1对体内细胞大小控制至关重要。我们还将使用老鼠遗传学来测试替代方案 P38应激激活蛋白激酶控制细胞大小的假说。总而言之，成功完成 这些目标将对理解细胞生长如何触发细胞分裂产生重大影响。这事很重要 因为它允许细胞控制自己的大小，这是细胞生理学的基础。

PROJECT SUMMARY This proposal aims to determine how cell growth triggers cell division, which is a fundamental question in cell and developmental biology. Its understanding will also greatly impact our knowledge of cancer, where this process is misregulated. It has long been known that cell growth triggers human cell division at the G1/S transition before DNA is replicated. But, although many key regulatory proteins linking cell growth to cell division are known, the molecular mechanisms mammalian cells use to control their size have remained poorly understood and have been based solely on the study of cells growing in culture. My laboratory recently made a breakthrough advance in understanding how growth triggers division. Contrary to expectations that growth would increase Cyclin D-Cdk4,6 activity, we found instead that cell growth dilutes the cell cycle inhibitor Rb to trigger division in cultured cells. Our discovery of the Rb dilution mechanism in cell culture raises three key questions which are the focus of this grant: 1. What is the molecular mechanism regulating Rb’s concentration dynamics that control cell size? 2. What is the function of Rb-based cell size control? 3. Do Rb dilution or other cell size control mechanisms link cell growth to cell division in vivo. We have begun to address the first question and our preliminary data indicate that the mechanism regulating the size-dependence of Rb concentration is translational. To further determine how this molecular mechanism works we will take an approach using reporters to identify the DNA-sequence element responsible and then the corresponding proteins regulating its function. To address the second question, we will take a mass spectrometry-based approach to measure how protein concentrations change with cell size across the proteome. Preliminary data indicate that proteins associated with senescence phenotypes increase in concentration in large cells. This suggests that cell size may be causal for senescence and cell size control functions to avoid this deleterious outcome. To address the final question to definitively test the Rb dilution and alternative models, we will perform a series of in vivo experiments. This is important because recent studies in cell culture have reported conflicting results about how animal cells control their size. To determine how animal cell growth triggers cell division in vivo, we propose to examine the mouse epidermis because it has a large population of proliferating stem cells whose division dynamics can be assayed using live- cell imaging. We will measure changes in keratinocyte cell size in a series of mouse lines in which the Rb family of genes has been conditionally deleted or over-expressed in the mouse epidermis. This will test our central hypothesis that Rb1 is crucial for cell size control in vivo. We will also use mouse genetics to test the alternative hypothesis that the p38 stress activated protein kinase controls cell size. Taken together, successful completion of these aims will have a big impact on understanding how cell growth triggers cell division. This is important because it allows cells to control their size, which is fundamental to cell physiology.