Functional characterization of newly identified cytoskeletal binding proteins in the control of actin myosin dynamics during chemotaxis.

定向细胞运动对胚胎发育和成人创伤愈合至关重要，需要适当控制才能实现这一目标。细胞通常受到其他细胞分泌的信号分子梯度的引导，这一过程被称为趋化性。趋化性是指细胞向化学吸引源的定向运动。因为这是一个非常重要的过程，科学家们已经对它进行了几十年的详细研究，一种模式生物已经被发现对这项研究特别有用，那就是一种社会性变形虫盘基骨虫。这是一种简单的生物，对小分子环状AMP表现出强烈的易于检测的趋化运动反应。细胞通过膜上的受体检测cAMP的梯度，并利用它们的皮质细胞骨架（相当于细胞的肌肉）来获得牵引力并产生导致细胞运动的力。在这个变形体运动的过程中，细胞需要在前面伸展膜突起并附着在表面上，然后在后面脱离表面，最后将整个细胞向前拉。这些过程和控制它们的机制在盘基骨柱细胞和人类免疫系统细胞之间都是高度保守的。为了执行所有这些步骤，每个细胞必须在空间和时间上精确地协调细胞骨架动力学。事实证明，这是一个非常复杂的过程，涉及几个信号通路和大量的调节因子和效应器并行地共同作用。由于这一过程的高度复杂性，以及在其调控中发生的高度冗余，目前还不可能在详细的机制水平上完全理解趋化性的所有方面，并且仍有许多悬而未决的问题。我们之前已经使用了一种基于尖端质谱的技术来测量构成细胞骨架的数百种蛋白质组成的快速变化，并控制其对化学引诱剂cAMP刺激的反应。我们已经分离并修改了数百种最有趣的成分的基因，使它们现在编码含有荧光标记的蛋白质，即所谓的绿色荧光蛋白（GFP）。这使我们能够在高度专业化和敏感的显微镜下看到这些蛋白质在活细胞中的定位，并在趋化运动期间跟踪它们在细胞中的定位变化。这告诉我们这些蛋白质很可能参与趋化过程。我们现在提议通过制造突变细胞来分析这些蛋白质的作用。对于每一种蛋白质，我们至少会制造出两种突变株，它们要么缺乏这种蛋白质，要么制造出过多的蛋白质。然后，我们将分析这些细胞在趋化过程中对cAMP的行为，这些行为的变化将告诉我们这种特殊蛋白质在这一过程中的作用。一旦我们确定了重要的组成部分，我们将进行实验，看看这些蛋白质是如何使用上述一些技术依次控制的。从长远来看，这将导致细胞如何检测cAMP梯度并修改细胞骨架以导致向化学引诱剂梯度方向运动的完整图景。一旦我们了解了趋化性是如何在一种简单生物盘基骨菌的细胞中详细地起作用的，我们就可以进行实验来证实这些过程在人类等高等生物的细胞中是相同的。这将对我们详细了解更复杂的过程产生重要影响，如胚胎发育、伤口愈合、免疫系统的功能以及许多重要疾病（如癌症）的检测和治疗。

Directed cell movement is critical for embryonic development, wound healing in adult life and needs to be properly controlled to achieve this. Often cells are guided by gradients of signalling molecules secreted by other cells and this process is known as chemotaxis. Chemotaxis is the directed cell movement towards the source of chemical attraction. Because it is such an important process scientists have been studying it in great detail for a few decades now and one model organism has been found to be particularly useful for this research, that is a social amoeba Dictyostelium discoideum. This is a simple organism that shows a strong easily detectable chemotactic movement response to a small molecule, cyclic AMP. Cells detect gradient of cAMP by receptors at the membrane and use their cortical cytoskeleton, a cellular equivalent of muscles, to gain traction and generate forces which lead to cell movement. During this process of amoeboid movement cells need to extend membrane protrusions at the front and attach them to the surface, then detach themselves from the surface at the back and finally pull the whole body of the cell forward. These processes and the machinery that controls them are highly conserved from Dictyostelium cells to cells of for instance the immune system in humans. In order to execute all these steps each cell has to precisely coordinate the cytoskeleton dynamics both in space and in time. This turns out to be a very complex process involving several signalling pathways and a huge number of regulators and effectors acting together in parallel. Due to very high complexity of this process and also high degree of redundancy occurring in its regulation it has not been yet possible to fully understand all the aspects of chemotaxis on a detailed mechanistic level and there remain many open questions. The research proposed here sets out to address some of these questionsWe have previously used a cutting edge mass spectrometry based technology to measure the rapid changes in the composition of the several hundred of proteins that make up the cytoskeleton and control its actions in response to stimulation with the chemo-attractant cAMP. We have isolated and modified the genes for more than hundred of the most interesting components so that they now code for proteins that contain a fluorescent label the so called Green Fluorescent Protein (GFP). This allows us to see the localisation of these proteins in living cells and follow changes in their localisation in the cell during chemotactic movement in a highly specialised and sensitive microscope. This has told us that these proteins are likely involved in the process of chemotaxis.We now propose to analyse the role of these proteins by making mutant cells. For every protein we will make at least two mutant strains that either lack or make too much of that protein. We will then analyse the behaviour of these cells during chemotaxis to cAMP and changes in the behaviour will tell us something about the role of this particular protein in the process. Once we have established important components we will perform experiments to see how these proteins are controlled in turn using some of the techniques described above. In the longer term this will lead to a complete picture of how cells detect gradient of cAMP and modify the cytoskeleton to result in movement in the direction of a chemo-attractant gradient. Once we understand how chemotaxis works in detail in cells of a simple organism Dictyostelium we can then perform experiments to confirm that these processes are the same in cells of higher organisms such as humans. This will have important consequences for our detailed understanding of more complex processes such as embryonic development, wound healing the functioning of the immune system and the detection and treatment of many important diseases such as cancer.