Determining how cell growth triggers cell division in epidermal stem cells

项目摘要 该提案旨在确定细胞生长如何触发细胞分裂，这是细胞生物学中的一个基本问题。 和发育生物学。它的理解也将极大地影响我们对癌症的认识， 过程是错误的。人们早就知道细胞生长会在G1/S期触发人类细胞分裂 在DNA复制之前，但是，尽管许多将细胞生长与细胞分裂联系起来的关键调节蛋白 尽管哺乳动物细胞用来控制其大小的分子机制是已知的， 它被理解并完全基于对培养中生长的细胞的研究。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。与预期相反， 增加细胞周期蛋白D-Cdk 4，6活性，我们发现细胞生长反而稀释了细胞周期抑制剂Rb， 在培养的细胞中分裂。我们在细胞培养中Rb稀释机制的发现提出了三个关键问题 这是该补助金的重点：1。调节Rb浓度动态的分子机制是什么 控制细胞大小的基因2.基于Rb的细胞大小控制的功能是什么？3.做Rb稀释或其他细胞大小 体内控制机制将细胞生长与细胞分裂联系起来。我们已经开始处理第一个问题， 初步数据表明，调节Rb浓度的尺寸依赖性的机制是平移的。 为了进一步确定这种分子机制是如何起作用的，我们将采用一种使用报告基因的方法来鉴定 负责的DNA序列元件，然后是调节其功能的相应蛋白质。解决 第二个问题，我们将采取基于质谱的方法来测量蛋白质浓度 随着细胞大小的变化而变化。初步数据表明与衰老相关的蛋白质 表型在大细胞中浓度增加。这表明细胞大小可能是衰老的原因 并且单元尺寸控制起作用以避免这种有害的结果。为了解决最后一个问题， Rb稀释和替代模型，我们将进行一系列的体内实验。这很重要因为 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。到 为了确定动物细胞生长如何在体内触发细胞分裂，我们建议检查小鼠表皮 因为它有大量的增殖干细胞，其分裂动力学可以用活的， 细胞成像我们将测量一系列小鼠品系中角质形成细胞大小的变化，其中Rb家族 在小鼠表皮中有条件地缺失或过表达。这将考验我们的中央 假设Rb 1对体内细胞大小控制至关重要。我们还将使用小鼠遗传学来测试替代方案 假设p38应激激活蛋白激酶控制细胞大小。综合来看，圆满完成 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。这很重要 因为它允许细胞控制它们的大小，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.