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：
10315927
负责人：
Jan M Skotheim
金额：
$ 42.54万
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/10315927
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 Size Cell division Cell physiology Cells Cellular biology Chemicals Code Conflict (Psychology)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 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 Proliferating Proteins Proteome Reporter Reporting Retinoblastoma Protein S Phase Series Testing Transitional Cell Translations Work base cell growth cell type epidermal stem cell expectation experimental study genetic regulatory protein in vivo in vivo imaging inhibitor/antagonist keratinocyte link protein live cell imaging live cell microscopy macrophage molecular modeling molecular size mouse genetics overexpression p38 Mitogen Activated Protein Kinase pathogen senescence 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.

项目摘要该建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了解，过程被误导。早就知道，细胞生长会触发G1/S处的人类细胞分裂复制DNA之前的过渡。但是，尽管许多关键的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理解并仅基于对培养细胞生长的研究。我的实验室最近做了突破性地了解增长如何触发分裂。与期望增长的期望相反增加细胞周期蛋白D-CDK4,6活性，我们发现细胞生长稀释了细胞周期抑制剂RB触发培养细胞的分裂。我们在细胞培养中发现RB部门机制的发现提出了三个关键问题这是该赠款的重点：1。那个控制单元格的大小？ 2。基于RB的单元尺寸控制的功能是什么？ 3。做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初步数据表明，计算RB浓度尺寸依赖性的机制是转化的。为了进一步确定这种分子机制的工作原理，我们将使用记者采取一种方法来识别负责的DNA序列元件，然后控制其功能的相应蛋白质。解决第二个问题，我们将采用基于质谱的方法来测量蛋白质浓度随着蛋白质组的细胞大小而变化。初步数据表明蛋白质与感应相关大细胞中浓度的表型增加。这表明细胞大小可能是感染的因果和细胞大小控制功能，以避免这种有害结果。解决最终问题以确定测试 RB稀释和替代模型，我们将执行一系列体内实验。这很重要，因为细胞培养的最新研究报道了有关动物细胞如何控制其大小的矛盾结果。到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具有大量增殖的干细胞，其分裂动态可以使用Live- 细胞成像。我们将在一系列RB家族的一系列小鼠系中测量角质形成细胞大小的变化在小鼠表皮中有条件地删除或过表达基因的基因。这将测试我们的中央假设RB1对于体内细胞尺寸控制至关重要。我们还将使用鼠标遗传学测试替代方案 p38应激激活蛋白激酶控制细胞大小的假设。两者合计，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。这很重要因为它允许细胞控制其大小，这是细胞生理基础的。