Mechanisms of restriction point response to dynamic growth factor signals

 描述(申请人提供)：在限制点(R)，哺乳动物细胞不可逆转地承诺分裂，并将继续分裂，即使生长因素被去除。R大多被认为是在DNA复制之前的G1末期的一个点，此时生长因子激活了激酶ERK，触发了CDK2活性的正反馈循环，使细胞周期的进展不可逆转。然而，最近的单细胞研究对这一模型提出了质疑，将R放在细胞周期的更早位置，就在有丝分裂之后，甚至在之前的细胞周期中。我们开发了一种单细胞实验，发现在原代细胞中，通过R的过程发生在第一次通过阈值水平的CDK2时的G1期。虽然我们的数据确定了R的阈值，但我们不知道生长因子信号如何决定细胞周期通过G1的速度。事实上，我们的初步数据和对文献的阅读表明，在G1早期至少存在一个额外的、目前尚未确定的控制点(R0)。目前对生长因子信号转导对R的影响的了解受到这样一个事实的限制，即以前只研究了少数生长因子的动态变化，如单步增加或单步减少或脉冲。为了克服低吞吐量手动介质交换的技术限制，我们创建了一个微流控平台，该平台集成了荧光成像和细胞外环境的自动时间控制。我们将使用我们的微流体平台来检测小鼠原代细胞的细胞周期进程，其中包含各种细胞周期进程的荧光报告。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对动态增殖信号做出反应，(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)确定细胞周期抑制物的动力学如何控制G1。癌症相关性：我们提出的对动态生长因子信号的增殖反应的系统研究将揭示G1内不同的调节点，超出先前特征的限制点。这将使您深入了解正常情况 癌症中的增殖控制及其错误调控。

 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.