Inferring the Forces Controlling Metaphase Kinetochore Oscillations by Reverse Engineering System Dynamics.
Inferring the Forces Controlling Metaphase Kinetochore Oscillations by Reverse Engineering System Dynamics.
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DOI：
10.1371/journal.pcbi.1004607
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发表时间：
2015-11
影响因子：
4.3
通讯作者：
Burroughs NJ
中科院分区：
文献类型：
--
作者：
Armond JW;Harry EF;McAinsh AD;Burroughs NJ
动粒是多蛋白质复合物，其介导姐妹染色单体与来自相应极的纺锤体微管束（称为动粒（K）-纤维）的物理偶联。这些附着有着丝粒的K-纤维产生推力和拉力，这些推力和拉力与极性喷射力（PEF）和弹性姐妹间染色质联合收割机结合以控制染色体运动。经典的减数分裂细胞实验中使用校准的微针测量染色体的近似失速力，但缺乏允许系统确定作用于活细胞中动粒的力的方法。在这里，我们报告的数学模型，可以安装（逆向工程）的高分辨率动粒跟踪数据的发展，从而估计模型参数，并允许我们间接计算（相对）力分量（K-纤维，弹簧力和PEF）在体内作用于个别姐妹动粒。我们将我们的方法应用于数千个人类动粒对轨迹，并在方向切换过程中报告时间力分布的不同签名。我们发现，K-纤维的力量是整个振荡的主导力量，和着丝粒弹簧最小，虽然它有最强的方向开关签名。在整个中期板块中也存在结构，朝着外围具有更陡的PEF势阱，并且伴随着板块厚度和振荡幅度的减小。这种数据驱动的逆向工程方法足够灵活，可以拟合更复杂的机械模型;因此，动粒动力学的数学模型可以首次在实验数据上进行彻底测试。未来的工作现在将能够绘制出单个蛋白质如何有助于基于kinetochorse的力的产生和感知。为了实现正确的细胞分裂，新复制的染色体必须以高保真度分离到子细胞中。这发生在有丝分裂中，在关键的中期阶段，染色体排列在一个假想的板上，称为中期板。染色体被称为动粒的蛋白质机器附着在一个结构支架上有丝分裂纺锤体，它由称为微管的动态纤维组成。在分裂中期对动粒的观察表明它们经历了一系列的向前和向后的运动。产生这种振荡运动的机械系统还不很清楚。通过在活细胞3D共聚焦显微镜中跟踪动粒并对其轨迹进行逆向工程，我们将作用在动粒上的力分解为三个主要的力生成分量。动粒动力学主要由K-纤维力，虽然随着时间的推移，在轻微的弹簧力的变化表明在控制方向切换的重要作用。此外，我们表明，力的强度可以在整个板内的细胞和细胞之间的空间变化。
Kinetochores are multi-protein complexes that mediate the physical coupling of sister chromatids to spindle microtubule bundles (called kinetochore (K)-fibres) from respective poles. These kinetochore-attached K-fibres generate pushing and pulling forces, which combine with polar ejection forces (PEF) and elastic inter-sister chromatin to govern chromosome movements. Classic experiments in meiotic cells using calibrated micro-needles measured an approximate stall force for a chromosome, but methods that allow the systematic determination of forces acting on a kinetochore in living cells are lacking. Here we report the development of mathematical models that can be fitted (reverse engineered) to high-resolution kinetochore tracking data, thereby estimating the model parameters and allowing us to indirectly compute the (relative) force components (K-fibre, spring force and PEF) acting on individual sister kinetochores in vivo. We applied our methodology to thousands of human kinetochore pair trajectories and report distinct signatures in temporal force profiles during directional switches. We found the K-fibre force to be the dominant force throughout oscillations, and the centromeric spring the smallest although it has the strongest directional switching signature. There is also structure throughout the metaphase plate, with a steeper PEF potential well towards the periphery and a concomitant reduction in plate thickness and oscillation amplitude. This data driven reverse engineering approach is sufficiently flexible to allow fitting of more complex mechanistic models; mathematical models of kinetochore dynamics can therefore be thoroughly tested on experimental data for the first time. Future work will now be able to map out how individual proteins contribute to kinetochore-based force generation and sensing. To achieve proper cell division, newly duplicated chromosomes must be segregated into daughter cells with high fidelity. This occurs in mitosis where during the crucial metaphase stage chromosomes are aligned on an imaginary plate, called the metaphase plate. Chromosomes are attached to a structural scaffold—the mitotic spindle, which is composed of dynamic fibres called microtubules—by protein machines called kinetochores. Observation of kinetochores during metaphase reveals they undergo a series of forward and backward movements. The mechanical system generating this oscillatory motion is not well understood. By tracking kinetochores in live cell 3D confocal microscopy and reverse engineering their trajectories we decompose the forces acting on kinetochores into the three main force generating components. Kinetochore dynamics are dominated by K-fibre forces, although changes in the minor spring force over time suggests an important role in controlling directional switching. In addition, we show that the strength of forces can vary both spatially within cells throughout the plate and between cells.