Mechanisms of restriction point response to dynamic growth factor signals

 描述（由申请人提供）：在限制点（R），哺乳动物细胞不可逆地进行分裂，即使去除生长因子也会继续分裂。R通常被认为是G1晚期DNA复制前的一个点，此时生长因子激活ERK激酶，触发Cdk2活性的正反馈环，使细胞周期的进展不可逆。然而，最近的单细胞研究对这一模型提出了质疑，因为R在细胞周期中的位置要早得多，就在有丝分裂之后，甚至在前一个细胞周期中。我们开发了一种单细胞测定，发现在原代细胞中，通过R的通道发生在G1中，在Cdk2活性的阈值水平的第一次通过。虽然我们的数据确定了R的阈值，但我们不了解生长因子信号传导如何决定细胞周期通过G1的进展速率。事实上，我们的初步数据和阅读的文献表明，存在至少一个额外的，目前未表征，控制点（R0）在早期G1。目前对生长因子信号传导对R的影响的理解受到以下事实的限制，即先前仅研究了少数生长因子的动态曲线，例如单步增加或单步减少或脉冲。为了克服低通量手动培养基交换的技术限制，我们创建了一个微流体平台，该平台将荧光成像与细胞外环境的自动化时间控制相结合。我们将使用我们的微流体平台来检查含有用于细胞周期进展的各种荧光报告基因的小鼠原代细胞中的细胞周期进展。这将使我们能够测试R0的分子基础和从G1到R的进展速度的特定假设。具体目标：（1）使用微流体来确定原代细胞如何响应动态增殖信号，（2）确定动态丝裂原活化蛋白激酶（MAPK）信号如何控制G1，以及（3）确定细胞周期抑制剂的动力学如何控制G1。癌症相关性：我们提出的系统研究动态生长因子信号的增殖反应，将揭示不同的点G1内的调控超出了以前的特点限制点。这将使我们了解正常的 增殖控制及其在癌症中的失调。

 DESCRIPTION (provided by applicant): At the restriction point (R), mammalian cells irreversibly commit to division and will go on to divide even if growth factors are removed. R has mostly been viewed as a point in late G1 just before DNA replication when growth factors activate the kinase ERK to trigger a positive feedback loop of Cdk2 activity that makes progression through the cell cycle irreversible. However, recent single-cell studies cast doubt on this model by placing R much earlier in the cell cycle just after mitosis or even within the previous cell cycle. We developed a single cell assay to find that in primary cells passage through R occurs in G1 at the first passing of a threshold level of Cdk2 activity. While our data identify the threshold for R, we do not understand how growth factor signaling determines the rate of cell cycle progression through G1. Indeed, our preliminary data and reading of the literature suggests the presence of at least one additional, currently uncharacterized, control point (R0) in early G1. Current understanding of the impact of growth factor signaling on R is limited by the fact that only a handful of dynamic profiles of growth factors, such as a single ste increase or a single step decrease or pulse, have previously been investigated. To overcome technical limitations of low-throughput manual media exchange, we have created a microfluidics platform that integrates fluorescence imaging with sharp automated temporal control of the extracellular environment. We will use our microfluidics platform to examine cell cycle progression in mouse primary cells containing a variety of fluorescent reporters for cell cycle progression. This will allow us to test specific hypotheses for the molecular basis of R0 and the rate of progression through G1 to R. Specific Aims: (1) Use microfluidics to determine how primary cells respond to dynamic proliferation signals, (2) Determine how dynamic mitogen activated protein kinase (MAPK) signals control G1, and (3) Determine how dynamics of cell cycle inhibitors control G1. Cancer relevance: Our proposed systematic investigation of the proliferative response to dynamic growth factor signals will reveal distinct points of regulation within G1 beyond the previously characterized restriction point. This will give insight into normal proliferative control and its misregulation in cancer.