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

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 questions
We 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.

Chemotaxis is crucial for many biological processes including development, the immune response and cancel metastasis. We still lack a detailed mechanistic description of key events and signalling pathways coupling signal detection by receptors at the membrane with the coordinated reorganisation of the actin-myosin cytoskeleton in the front and the back of the cell driving cell movement. Dictyostelium discoideum is a leading model system in which to investigate these processes due to its well-developed molecular genetics that allows efficient manipulation of key components through rapid knock-out and knock-in studies and analysis methods. We have utilised SILAC based quantitative proteomic methods to analyse the chemoattractant (cAMP) induced cytoskeletal dynamics on a systems wide scale. These experiments allowed us for the first time to analyse dynamic changes in protein composition of the entire cytoskeleton and associated proteins during the chemotactic response in a quantitative manner. We detected a few hundred cytoskeletal and associated proteins most of which were showing rapid changes in incorporation and association with the cytoskeleton after cAMP stimulation. Many of these components, among them many regulators of the Rac family of small GTPases have clear orthologs in higher organism, but are as yet uncharacterised. Stimulation dependent translocation was validated by in vivo imaging for 120 GFP tagged components. Around 20 of the most interesting uncharacterised proteins, showing distinct kinetics and cellular localisation will be further investigated to determine their function in the chemotactic response. These studies will involve the detailed analysis of the phenotypes in cytoskeletal dynamics, motility and chemotactic movement of knockout, knockin and over-expression strains. We will furthermore identify interaction partners through SILAC based mass spec methods that will help to place these components in distinct chemotactic signalling pathways.

The cytoskeleton is highly dynamic and possible the largest machinery in the cell and is responsible for performing all the work needed for essential processes such as cell division, cell shape changes, integration of cells in tissue and cell motility during development and adult life as well as the coordination of many cell internal processes such as transport, endocytosis, pinocytosis and receptor internalisation and recycling. These function as are essential to life and need to be carefully controlled in space and time. As a result the cytoskeleton is the target for many of the known signalling pathways. The research proposed here is directed towards obtaining further insights in the control and regulation of the actin myosin cytoskeleton dynamics responsible for chemotactic cell movement. Directed cell movement is essential for development and in adult life, in functioning of the immune system and tissue regeneration. Misregulation of motility results in many congenital defects and diseases in adult life. Therefore understanding the processes that control cell motility contribute to "basic bioscience underpinning health" one of the BBSRC's strategic priorities. It also covers "exploiting new ways of working", especially "data driven biology" and "systems approaches to the biosciences". The result obtained here will provide inputs into "synthetic biology" approaches in the future, with the aim of engineering migration.
The research to be conducted here could also have important practical consequences in the not so distant future. It can result in better understanding and diagnosis of various congenital defects, disorders of the immune system and result in improvement treatments of disorders of the immune system and metastatic diseases such as cancer. The knowledge obtained here will also be valuable for the rational use of stem cells in regenerative medicine. Furthermore we will develop methods, especially in rapid life imaging and motility assays that are transferable to other experimental systems and therefore will have impact on the research performed by other researchers. Finally the proposed project will allow the further development of the scientific career of Dr Grzegorz Sobzcyk, especially in areas of advanced proteomics, large scale data analysis and development of novel motility assays and optical methods to assay cell behaviours. The Life Sciences sector has an important economic impact in Dundee, contributing around 16% of the city's GDP. A range of activities and organisations in the city connect scientists with the public. In recognition of the economic and social impact of these interactions, the College of Life Sciences won the BBSRC "Excellence with Impact" Award in 2011.