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

点击翻译按钮获取中文摘要

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.