Determining how cell growth triggers cell division in epidermal stem cells

确定细胞生长如何触发表皮干细胞的细胞分裂

基本信息

批准号：
10636863
负责人：
Jan M Skotheim
金额：
$ 41.71万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-15 至 2026-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10636863
关键词：
3&apos Untranslated Regions 5&apos Untranslated Regions Address Anabolism Animals B-Lymphocytes Biological Assay Blood capillaries Cell Aging Cell Culture Techniques Cell Cycle Cell Cycle Proteins Cell Proliferation Cell Size Cell division Cell physiology Cells Cellular biology Chemicals Code Coupling Cultured Cells Cyclin D1 DNA DNA Sequence Data Dependence Developmental Biology Elements Epidermis Erythrocytes G1/S Transition G2 Phase Gene Family Genetic Grant Growth Human Knowledge Laboratories Link Macrophage Malignant Neoplasms Mammalian Cell Mammals Mass Spectrum Analysis Measures Messenger RNA Modeling Molecular Mus Nuclear Proteins Outcome Pathology Phase Phenotype Physiological Pilot Projects Population Process Proteins Proteome Reporter Reporting Retinoblastoma Protein S phase Series Sorting Testing Translations Work cell growth cell type epidermal stem cell expectation experimental study genetic regulatory protein in vivo in vivo imaging inhibitor keratinocyte link protein live cell imaging live cell microscopy molecular modeling molecular size mouse genetics overexpression p38 Mitogen Activated Protein Kinase pathogen senescence stem cell proliferation stem cells stress activated protein kinase two photon microscopy two-photon

项目摘要

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.

项目概要该提案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它的理解也将极大地影响我们对癌症的认识。人们早就知道细胞生长会触发 G1/S 期的人类细胞分裂。但是，尽管许多关键调节蛋白将细胞生长与细胞分裂联系起来。众所周知，哺乳动物细胞用来控制其大小的分子机制仍然很差我的实验室最近做了一个研究，并完全基于对培养细胞的研究。在理解增长如何引发分裂方面取得了突破性进展。增加 Cyclin D-Cdk4,6 活性，我们发现细胞生长反而会稀释细胞周期抑制剂 Rb 以触发我们对细胞培养中 Rb 稀释机制的发现提出了三个关键问题。本次资助的重点： 1. 调节 Rb 浓度动态的分子机制是什么控制细胞大小？ 2. 基于 Rb 的细胞大小控制有何作用？ 3. 是否进行 Rb 稀释或其他细胞大小？控制机制将细胞生长与体内细胞分裂联系起来我们已经开始解决第一个问题和我们的问题。初步数据表明，调节 Rb 浓度大小依赖性的机制是平移的。为了进一步确定这种分子机制如何发挥作用，我们将采取一种方法，利用生产者来识别负责的DNA序列元件以及调节其功能的相应蛋白质。第二个问题，我们将采用基于质谱的方法来测量蛋白质浓度初步数据表明，蛋白质组中的蛋白质随细胞大小而变化。大细胞中的表型浓度增加，这表明细胞大小可能是衰老的原因。和细胞大小控制功能可以避免这种有害结果。 Rb 稀释和替代模型，我们将进行一系列体内实验，因为这很重要。最近的细胞培养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。为了确定动物细胞生长如何触发体内细胞分裂，我们建议检查小鼠表皮因为它有大量的增殖干细胞，其分裂动态可以使用活细胞进行测定我们将测量一系列含有 Rb 家族的小鼠品系的角质细胞大小的变化。的基因已在小鼠表皮中被有条件地删除或过度表达，这将测试我们的中枢。 Rb1 对于体内细胞大小控制至关重要的假设我们还将使用小鼠遗传学来测试替代方案。综上所述，p38应激激活蛋白激酶控制细胞大小的假设成功完成。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。因为它允许细胞控制它们的大小，这是细胞生理学的基础。