The Role and Regulation of Tiam1-Rac Signalling in Bipolar Spindle Assembly

在细胞分裂过程中，我们的两组相同的染色体需要精确地分裂成两个子细胞。这个过程依赖于有丝分裂的双极纺锤体，这是一种由微管（动态细胞骨架结构）组成的结构，它捕获染色体。纺锤体的形成涉及两个中心体，它们是微管成核的组织中心。当细胞进入有丝分裂时，中心体分开形成纺锤体的方式受到高度调节。科学家们之前已经证明了一种蛋白质，Eg5，对这种分离非常重要。靶向这种蛋白质的药物完全阻止中心体分离，导致细胞以单极体纺锤体进行有丝分裂。我们最近发现，一种蛋白（Tiam1）及其底物（Rac）在有丝分裂中对调节癌细胞的迁移起重要作用，对调节中心体分离过程也很重要。在纺锤体形成过程中，Tiam1和Rac产生的力抵消了Eg5的力。我们已经证明，这种力量的平衡对于染色体被微管有效地捕获是必要的。我们还发现Tiam1与MPF相互作用，MPF是一种重要的有丝分裂复合体（也调节Eg5的功能），MPF对Tiam1的调节对于Tiam1调节中心体分离的功能很重要。然而，我们目前还不知道Tiam1和Rac在这一过程中的下游过程，很可能涉及其他一些未知的蛋白质。了解细胞分裂过程中的分子机制对癌症研究至关重要。首先，我们知道，准确分裂的失调可以通过重要基因的丢失或获得而导致癌症。其次，许多目前的治疗针对细胞分裂过程，因为癌细胞比正常细胞分裂得更快，所以知道哪些蛋白质调节这一过程使我们能够预测癌症对某些化疗的反应。Tiam1和Rac产生的作用力意味着，当其中一种蛋白质缺失或受到抑制时，细胞可以更容易地“逃脱”Eg5的抑制。目前，Eg5抑制剂正处于癌症治疗的临床试验中，因此更仔细地研究这种信号通路的机制对于能够预测癌症对这种新型癌症疗法的反应是很重要的。我们认为Tiam1和Rac通过调节微管对中心体分离产生影响的方式，也意味着它们可以改变细胞对另一类癌症疗法的反应，即以微管为目标杀死癌细胞。我们计划通过在癌细胞中重复我们的实验来进一步研究这一点，看看Tiam1和Rac是否可以作为这些药物治疗的生物标志物。由于这种信号通路对肿瘤发生和癌症治疗的潜在重要性，我们的目标是进一步表征信号通路，我们之前的工作意味着我们已经有了许多可用的工具来实现这一目标。这个项目的一部分包括更密切地观察Tiam1和Rac对微管的调节。这项工作的另一个重要方面是确定与Tiam1和Rac一起调节中心体分离的蛋白质。我们可以采取两种不同的方法；首先，我们可以使用我们拥有的工具来启动新的潜在相互作用蛋白质的筛选，其次，我们可以使用候选方法来单独测试我们以前的实验或文献中的候选蛋白质，这些候选蛋白质似乎可能涉及。这项工作不仅将进一步加深我们对有丝分裂中中心体调节的重要过程的理解，而且，如上所述，可能对肿瘤发生和当前的癌症治疗具有重要意义。

During cell division our two identical sets of chromosomes need to be divided accurately into two daughter cells. This process relies on the mitotic bipolar spindle, a structure composed of microtubules (dynamic cytoskeletal structures) which capture the chromosomes. Formation of the spindle involves two centrosomes, the organising centres for microtubule nucleation. The way the centrosomes move apart to form the spindle when cells enter mitosis is very highly regulated. Scientists have previously shown that one protein in particular, Eg5, is very important for this separation. Drugs which target this protein completely stop the centrosomes separating and cause cells to arrest in mitosis with 'monopolar' spindles. We have recently found that a protein (Tiam1) and its substrate (Rac), which were previously known to be important in regulating the migration of cancer cells, are also important for regulating this centrosome separation process in mitosis. Tiam1 and Rac produce a force which counteracts the force of Eg5 during spindle formation. We have shown that this balance of forces is necessary to allow the chromosomes to become efficiently captured by the microtubules. We have also found that Tiam1 interacts with the MPF, an important mitotic complex (which also regulates the function of Eg5), and that regulation of Tiam1 by MPF is important for the function of Tiam1 in regulating centrosome separation. However, we currently do not know the processes downstream of Tiam1 and Rac in this process, and it is likely that a number of other, unknown proteins are involved. Understanding the molecular mechanisms involved in the process of cell division is extremely important for cancer research. Firstly, it is known that de-regulation of accurate division can cause cancer by the loss or gain of important genes. Secondly, many current treatments target the process of cell division because cancer cells divide more rapidly than normal cells, so knowing which proteins regulate this process allow us to predict how cancers will respond to certain chemotherapies. The force produced by Tiam1 and Rac means that when one of these proteins is absent, or inhibited, cells can more readily 'escape' inhibition of Eg5. Currently, Eg5 inhibitors are in clinical trials for cancer treatment, so looking more closely at the mechanisms of this signalling pathway is important for being able to predict how cancers will respond to this new class of cancer therapeutics. The way that we think Tiam1 and Rac exert their effect on centrosome separation, namely by regulating microtubules, also means they can alter cells' responses to another class of cancer therapeutics, that target the microtubules to kill cancer cells. We plan to investigate this further, by repeating our experiments in cancer cells to see whether Tiam1 and Rac could be a biomarker for treatment with these drugs.Because of the potential importance of this signalling pathway for tumourigenesis and cancer therapy, we aim to further characterise the signalling pathway, and our previous work means that we already have a number of tools available to achieve this. Part of this project involves looking more closely at the regulation of microtubules by Tiam1 and Rac. Another important aspect of this work is to identify the proteins which work together with Tiam1 and Rac to regulate centrosome separation. We can take two different approaches to this; firstly, we can use the tools we have to initiate a screen for new potential interacting proteins, and secondly we can use a candidate approach to individually test candidates from our previous experiments, or the literature, which seem likely to be involved. This work will not only further our understanding of the important process of centrosome regulation in mitosis, but, as described above, may have important implications for tumourigenesis and current cancer therapies.