Investigating the role of actin dynamics on receptor clustering and oligomerisation.

Closing date 31 March, unless filled beforehand

Project Overview and Aims:

One of the major ways in which cell-cell communication is mediated is through the binding of chemical messengers to cell surface proteins, called receptors. The way in which receptors relay their signals to the cell cytoplasm and nucleus is highly dependent on how they are organised within the cell membrane. The actin cytoskeleton is critical for this organisation and plays a key role in receptor clustering and oligomerisation, as well as potentially in the organisation of how receptors interact with their signalling proteins. Recently a very dynamic fine F-actin network has been identified at the cell cortex which allows for rapid movement of receptors and actively drives receptor clustering, with actin polymerisation regulated by the Arp2/3 and formin proteins.

This project will use a combination of advanced microscopy techniques such as single molecule localisation microscopy (SMLM), live cell super-resolution imaging and fluorescence correlation spectroscopy (FCS) to visualise this F actin and it’s dynamics with high-resolution. Alongside this we will monitor receptor dynamics and diffusion to describe how the cytoskeleton influences receptor behaviour and to investigate the receptor specific actin binding proteins and signalling pathways that drive this behaviour. In this work we will focus on two major classes of receptors: G protein coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs).

Project Plan:

The project will use gene editing techniques (CRISPR-Cas9) to fluorescently tag receptors at natural levels of expression in a Jurkat T cell line which naturally express several cell surface receptors linked to the cytoskeleton (GPCRs – CXCR4 chemokine, Adenosine- A2A and -A2B; RTKs – EGFR). By expressing fluorescent versions of these receptors along with a fluorescent actin the interaction between the receptors and cytoskeleton can be visualised. The study will also utilise small molecule inhibitors of actin polymerisation and fluorescent receptor ligands to understand how this interaction is modified by ligand binding or cytoskeletal disruption.

Techniques and project organisation

This will be a multi-disciplinary project giving the student experience over a wide number of techniques including cell culture, molecular biology, CRISPR-Cas9 and standard biochemical pharmacological techniques. However, the main focus will be on microscopy and advanced imaging approaches to looking at cytoskeletal and receptor organisation in the cell.

The project will be split between the Medical School in Birmingham and the School of Life Sciences in Nottingham and will take advantage of state of the art systems and imaging expertise. Both sites have newly refurbished imaging facilities with Nottingham having expertise in FCS and receptor dynamics and Birmingham in SMLM and actin dynamics. Both imaging facilities are supported by microscope and image analyst specialists and have access to advanced computing facilities. The project will be initially based mainly in Birmingham, but with increasing periods of time spent in Nottingham for the pharmacological and FCS-based aspects in years 2 and 3. However, the student will meet with both supervisors on a regular basis to ensure that the project is successful.

The project is one of six that has been funded through the Centre of Membrane Proteins and Receptors (COMPARE: http://www.birmingham-nottingham.ac.uk/compare/index.aspx) that focus on receptor clustering. The six studentships will form a doctoral training programme that will provide additional training and opportunities for collaboration.

Applications should include a letter explaining why this project is of interest to the applicant along with a CV and the names of two referees. They should be sent to BR-BN-COMPARE [Email Address Removed] by 31 March 2018.