Department of Biochemistry, University of Colorado-Boulder, Boulder, CO 80303.
BioFrontiers Institute, University of Colorado-Boulder, Boulder, CO 80309.
Proc Natl Acad Sci U S A. 2018 Aug 28;115(35):E8219-E8227. doi: 10.1073/pnas.1722446115. Epub 2018 Aug 15.
The Restriction Point was originally defined as the moment that cells commit to the cell cycle and was later suggested to coincide with hyperphosphorylation of the retinoblastoma protein (Rb). Current cell cycle models posit that cells exit mitosis into a pre-Restriction Point state, where they have low cyclin-dependent kinase (CDK) activity and hypophosphorylated Rb; passage through the Restriction Point then occurs in late G1. Recent single-cell studies have challenged the current paradigm, raising questions about the location of the Restriction Point and the notion that cells exit mitosis into a pre-Restriction Point state. Here, we use a variety of single-cell techniques to show that both noncancer and cancer cells bifurcate into two subpopulations after anaphase, marked by increasing vs. low CDK2 activity and hyper- vs. hypophosphorylation of Rb. Notably, subpopulations with hyper- and hypophosphorylated Rb are present within minutes after anaphase, delineating one subpopulation that never "uncrosses" the Restriction Point and continues cycling and another subpopulation that exits mitosis into an uncommitted pre-Restriction Point state. We further show that the CDK inhibitor p21 begins rising in G2 in mother cells whose daughters exit mitosis into the pre-Restriction Point, CDK2 state. Furthermore, degradation of p21 coincides with escape from the CDK2 state and passage through the Restriction Point. Together, these data support a model in which only a subset of cells returns to a pre-Restriction Point state after mitosis and where the Restriction Point is sensitive to not only mitogens, but also inherited DNA replication stress via p21.
限制点最初被定义为细胞进入细胞周期的时刻，后来有人提出它与视网膜母细胞瘤蛋白（Rb）的过度磷酸化相一致。目前的细胞周期模型假设细胞从有丝分裂进入限制点前状态，此时它们的细胞周期蛋白依赖性激酶（CDK）活性较低，Rb 呈低磷酸化状态；然后在 G1 晚期通过限制点。最近的单细胞研究对当前的范例提出了挑战，引发了关于限制点位置的问题，以及细胞从有丝分裂进入限制点前状态的概念。在这里，我们使用各种单细胞技术表明，有丝分裂后期，非癌细胞和癌细胞都会分叉成两个亚群，其特征是 CDK2 活性增加与降低以及 Rb 的过度磷酸化与低磷酸化。值得注意的是，在有丝分裂后期几分钟内，就存在 Rb 高磷酸化和低磷酸化的亚群，划分为一个从未“交叉”限制点并继续循环的亚群和另一个进入未承诺的限制点前状态的亚群。我们进一步表明，CDK 抑制剂 p21 在母细胞的 G2 中开始上升，其女儿进入限制点前的 CDK2 状态。此外，p21 的降解与从 CDK2 状态逃逸和通过限制点一致。这些数据共同支持这样一种模型，即只有一部分细胞在有丝分裂后回到限制点前状态，并且限制点不仅对有丝分裂原敏感，而且对通过 p21 遗传的 DNA 复制应激也敏感。
