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

新鉴定的细胞骨架结合蛋白在趋化过程中控制肌动蛋白肌球蛋白动力学的功能表征。

• 批准号: BB/L00271X/1
• 负责人: Kees Weijer
• 金额: $ 69.09万
• 依托单位: University of Dundee
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2013
• 资助国家: 英国
• 起止时间: 2013 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FL00271X%2F1
• 关键词: Functional; characterization; newly; identified; cytoskeletal

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.

定向细胞运动对于胚胎发育、成年期伤口愈合至关重要，需要适当控制以实现这一目标。细胞通常由其他细胞分泌的信号分子梯度引导，这个过程被称为趋化性。趋化性是细胞朝向化学吸引源的定向运动。因为这是一个如此重要的过程，科学家们已经对其进行了数十年的详细研究，并且发现一种模式生物对这项研究特别有用，那就是社会阿米巴Dictyosteelium discoideum。这是一种简单的生物体，对小分子环AMP表现出很强的易检测的趋化运动反应。细胞通过膜上的受体检测cAMP的梯度，并使用它们的皮质细胞骨架（一种相当于肌肉的细胞）来获得牵引力并产生导致细胞运动的力。在这个变形虫运动的过程中，细胞需要在前面延伸膜突起并将它们附着在表面上，然后在后面将它们从表面分离，最后将整个细胞向前拉动。这些过程和控制它们的机制从网骨藻细胞到例如人类免疫系统的细胞是高度保守的。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，涉及多个信号通路和大量的调节因子和效应因子并行作用。由于这一过程非常复杂，而且在其调节过程中存在高度冗余，因此尚不可能在详细的机械水平上完全理解趋化性的所有方面，并且仍然存在许多悬而未决的问题。这里提出的研究旨在解决其中的一些问题。我们以前使用了基于质谱的尖端技术来测量组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上百种最有趣的成分的基因，使它们编码含有荧光标记的蛋白质，即所谓的绿色荧光蛋白（GFP）。这使我们能够看到这些蛋白质在活细胞中的定位，并在高度专业化和灵敏的显微镜下观察它们在趋化运动过程中在细胞中的定位变化。这告诉我们这些蛋白质可能参与趋化过程，我们现在建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，我们将至少制造两种突变株，它们要么缺乏这种蛋白质，要么制造太多这种蛋白质。然后，我们将分析这些细胞在对cAMP趋化过程中的行为，行为的变化将告诉我们一些关于这种特定蛋白质在这个过程中的作用。一旦我们建立了重要的组成部分，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的梯度并修改细胞骨架以导致在化学引诱物梯度方向上的移动的完整画面。一旦我们详细了解了趋化性在简单生物Dictyosteopathy细胞中的作用，我们就可以进行实验来证实这些过程在高等生物如人类的细胞中是相同的。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的检测和治疗，如癌症。

项目成果

Oscillatory cAMP cell-cell signalling persists during multicellular Dictyostelium development.

振荡 cAMP 细胞间信号传导在多细胞盘基网柄菌发育过程中持续存在。

• DOI: 10.1038/s42003-019-0371-0
• 发表时间: 2019
• 期刊: Communications biology
• 影响因子: 5.9
• 作者: Singer G

Modelling cell movement, cell differentiation, cell sorting and proportion regulation in Dictyostelium discoideum aggregations.

对盘基网柄菌聚集体中的细胞运动、细胞分化、细胞分选和比例调节进行建模。

• DOI: 10.1016/j.jtbi.2015.01.042
• 发表时间: 2015
• 期刊: Journal of theoretical biology
• 影响因子: 2
• 作者: Pineda M

Chemotaxis overrides Barotaxis during Directional Decision-Making in Dictyostelium discoideum

• DOI: 10.1101/2020.01.14.904748
• 发表时间: 2020-01
• 期刊: bioRxiv
• 作者: Y. Belotti;D. Mcgloin;C. Weijer

SILAC-based proteomic quantification of chemoattractant-induced cytoskeleton dynamics on a second to minute timescale.

• DOI: 10.1038/ncomms4319
• 发表时间: 2014-02-26
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Sobczyk, Grzegorz J.;Wang, Jun;Weijer, Cornelis J.
• 通讯作者: Weijer, Cornelis J.

Effects of spatial confinement on migratory properties of Dictyostelium discoideum cells.

• DOI: 10.1080/19420889.2021.1872917
• 发表时间: 2021-01-28
• 期刊: Communicative & integrative biology
• 作者: Belotti Y;McGloin D;Weijer CJ
• 通讯作者: Weijer CJ