Kinetochore life-histories: understanding the mechanical events that ensure error-free chromosome segregation

现代细胞生物学的一个基本挑战是理解复杂的行为是如何从分子机器群体中产生的，分子机器工作在接近热能水平的水平，从而使它们的行为具有显著的随机成分，同时在全球通信网络的背景下独立工作。由于这些系统的体积很小，使用它们带来了巨大的挑战；然而，光学显微镜的最新进展使人们能够以前所未有的空间和时间分辨率对这些系统进行观察和量化。我们感兴趣的生物系统是如何在细胞分裂过程中分离染色体的。人类是由一个受精细胞--受精卵构成的。每个细胞包含46条染色体--遗传物质(DNA)的包装，它为细胞如何工作以及如何构建人类提供了指令。细胞分裂产生两个几乎相同的子细胞。染色体在分裂前被复制，细胞分裂的一个关键部分是准确分离这些复制的染色体，使每个女儿收到且只有一个副本。细胞不能获得一套完整的46条染色体是多种人类疾病的原因，包括癌症和唐氏综合症。在细胞分裂的机械过程中，大部分时间都花在将成对的染色体(原始和复制)重新定位到赤道平面上的保持模式上，然后再将成对的染色体拉到细胞的两端。显然，弄清楚染色体分离是如何工作的是至关重要的。染色体分离是一个机械的空间过程。为了移动染色体，细胞利用被称为微管的分子电缆，可以生长和收缩。每条染色体都有一个名为着丝粒的“钩”，它连接到这些电缆的两侧(横向连接)和这些电缆的末端。动粒是一种非常通用的智能机器，包括传感器和马达，允许它确定它是如何连接到微管上的，以及它的配对姐妹如何连接，做出一系列明智的决定，以便将配对的染色体重新定位到赤道平面。因此，着丝粒是决定染色体何时何地移动的“控制中心”。但是，动毛虫是如何做到这一点的呢？Kintochore为什么和如何做出决定，并使用什么信息作为输入？我们计划进行的实验将有助于回答这些令人兴奋和有趣的问题，从而加深我们对染色体在细胞分裂过程中如何忠实地分离为子细胞的理解。我们将使用最先进的成像技术(显微镜)来观察染色体在活细胞中的运动。然后，我们将使用数学建模和复杂的统计技术(称为逆向工程)来根据数据确定该模型的参数(一个人根据观察确定系统必须是什么)。这将使我们能够推断动粒使用什么信号来调节附着的微管。这将包括作用在动粒以及动粒内部拉伸和旋转上的力，我们之前的研究表明，动粒具有类似髋关节的结构，这可能防止在冲力下附件的断裂。我们的模型将与我们的数据一样好；因此，我们将使用各种技术来扰乱系统(移除或拆卸某些组件，从而改变行为)，从而在一系列不同的情况下‘测试’我们的模型。通过这些技术，我们将实现更好的生物学理解。

A fundamental challenge in modern cell biology is to understand how complex behaviours emerge from populations of molecular machines, machines that work close to the thermal energy level, thereby giving their behaviour a significant random component, whilst working independently within the context of a global communication network. Working with such systems poses significant challenges given their small size; however recent advances in light microscopy has enabled these systems to be observed and quantified with unprecedented spatial and time resolution. The biological system we are interested in is how chromosomes are separated during cell division. Human beings are built from a single fertilised cell, the zygote. Each cell contains 46 chromosomes - the packages of genetic material (DNA), which provide the instructions for how a cell should work and how to build a human. Cells divide generating two near identical daughter cells. The chromosomes are copied prior to division and a key part of cell division is the accurate separation of these replicated chromosomes such that each daughter receives 1 and only 1 copy. Failure of a cell to receive a complete set of 46 chromosomes is a cause of multiple human diseases, including cancers and Down's syndrome. During the mechanical process of cell division, most of the time is taken up with relocating the paired chromosomes (original and copy) into a holding pattern at the equatorial plane, prior to pulling the pairs apart to either end of the cell. Clearly it is vital that we work out how chromosome separation works.Chromosome separation is a mechanical spatial process. To move a chromosome the cell makes use of molecular cables called microtubules that can grow and shrink. Each chromosome has a "hook" called the kinetochore, which attach to these cables, both on their sides (lateral attachment) and to the ends of these cables. The kinetochore is an extremely versatile and 'intelligent' machine, comprising sensors and motors that allow it to determine how it is attached to microtubules and how its paired sister is attached, making a sequence of informed decisions so that the paired chromosomes are relocated to the equatorial plane. The kinetochore is thus the "control centre" that decides when and where a chromosome moves. But, how does the kinetochore do this? Why and how do kinetochores make decisions, and using what information as input? The experiments that we propose to carry out will help answer these exciting and intriguing question and therefore advance our understanding of how chromosomes are faithfully separated into daughter cells during cell division.We will use state-of-the-art imaging technology (microscopes) to observe how chromosomes move in living human cells. We will then use mathematical modelling and sophisticated statistical techniques (called reverse engineering) to determine the parameters of that model from the data (one engineers what the system must be from the observations). This will allow us to infer what cues the kinetochore is using to regulate the attached microtubules. This will include forces acting on the kinetochores and internal stretch and rotation of the kinetochore, our previous study showing that the kinetochore has a structure similar to a hip joint which potentially prevents breakage of the attachment under impulse forces. Our model will only be as good as our data; thus we will use a variety of techniques to perturb the system (remove or knock-down certain components, thereby changing behaviour), and thus 'road-test' our model through a range of different situations. Through such techniques we will achieve greater biological understanding.