Analysis of lipid and lipid-anchored protein organization at the T-cell surface using novel super-resolution (STED-FCS) microscopy
The organization of the resting T-cell surface is presently a focus of intense interest for molecular immunologists . One view is that surface proteins are generally monomeric and freely diffusing, whereas it is alternatively suggested that movement is constrained to protein islands, or that important components of the signalling machinery are kept apart from one another, e.g. in order to prevent unwanted cellular activation.
An additional issue concerns the extent to which membrane lipids form organized domains, either spontaneously or in the immediate vicinity of surface proteins, and how the lipids are involved in the signalling process, if at all . Unfortunately, many details of protein/lipid organizations and interaction dynamics cannot accurately be determined in the living cell because of the limited spatial resolution of far-field optical fluorescence microscopy. While this imaging technique is currently probably the most valuable tool for directly investigating the living cell with minimal invasion, similar objects closer together than approximately 200 nm cannot be distinguished and details of molecular organization and dynamics on (macro)molecular scales cannot be recovered directly. A remedy to this is recently developed super-resolution optical microscopy or nanoscopy: approaches such as STED (stimulated emission depletion microscopy) have now evolved into superior tools for investigating cellular dynamics at the nanoscale [3,4]. Specifically the combination of STED with fluorescence correlation spectroscopy (STED-FCS) has recently allowed unique insights into molecular plasma membrane dynamics and organization such as the potential formation of nanodomains (or lipid “rafts”) [5,6].
One of the key proteins at the heart of the remaining questions on the molecular organization and dynamics of the resting T-cell surface is the Src-type tyrosine kinase, Lck, which initiates signalling in T-cells by phosphorylating the T-cell receptor (TCR). Lck is of considerable intrinsic interest firstly because its activity needs to be constrained in order to prevent uncontrolled T-cell activation, and secondly because it is an unusual surface component insofar as it associates with the membrane via two types of lipid-type anchors i.e. myristoyl and palmitoyl groups, and it is unclear how or whether this affects the distribution and/or function of this kinase. In the case of the TCR, it has been suggested that receptor ligation induces changes in the lipid environment around the receptor, which facilitates the access of downstream signalling molecules, including kinases. A third point of interest is that, in contrast to other Src kinases, Lck associates with the co-receptor CD4, which is an integral membrane protein. It is possible that the lipid micro-environment and dynamics of CD4-bound and -unbound Lck diffusion differ. We propose to characterize the organization and interaction dynamics of a lipid-anchored protein at the T-cell surface, i.e. myristoylated and palmitoylated Lck, both in relation to different lipids, and in comparison to other components of the signalling machinery of the T-cell including CD4, using STED(-FCS) microscopy. We expect these novel experiments to highlight, in thus far unprecedented detail, the organization of key signalling proteins at the T-cell surface, and to create a critical framework for understanding receptor triggering.
This project will be based in the MRC Human Immunology Unit at the Weatherall Institute of Molecular Medicine, with access to state-of-the-art facilities. The project provides an opportunity for training in a broad range of different techniques, such as cell culture, molecular biology, and microscopy, specifically including the unique STED-FCS super-resolution microscopy technique. The disclosure of novel details of T-cell activation is an important line of basic immunological research that may translate into new approaches of modulating the immune response during infection and may pave the way to new vaccine adjuvants. Close collaboration with many scientists will be required.
As well as the specific training detailed above, students will have access to a wide-range of seminars and training opportunities through the many research institutes and centres based in Oxford. Students are also able to attend the Methods and Techniques course run by the MRC Weatherall Institute of Molecular Medicine. This course runs through the year, ensuring that students have the opportunity to build a broad-based understanding of differing research techniques.
Generic skills training is offered through the Medical Sciences Division's Skills Training Programme. This programme offers a comprehensive range of courses covering many important areas of researcher development: knowledge and intellectual abilities, personal effectiveness, research governance and organisation, and engagement, influence and impact. Students are actively encouraged to take advantage of the training opportunities available to them.
The Department has a successful mentoring scheme, open to graduate students, which provides an additional possible channel for personal and professional development outside the regular supervisory framework. We hold an Athena SWAN Silver Award in recognition of our efforts to support the careers of female students and staff.
|1||J.R. James, J. McColl, M.I. Oliveira, P.D. Dunne, E. Huang, A. Jansson, P. Nilsson, D.L. Sleep, C.M. Gonçalves, S.H. Morgan, J.H. Felce, R. Mahen, R.A. Fernandes, A.M. Carmo, D. Klenerman, S.J. Davis. The T cell receptor triggering apparatus is composed of monovalent or monomeric proteins . J Biol Chem 286, 31993-200 (2011).|
|2||K. Simons, M.J. Gerl. Revitalizing membrane rafts: new tools and insights. Nature Rev Mol Cell Biol 11, 688-99 (2010).|
|3||C. Eggeling, K.I. Willig, F.J. Barrantes. STED microscopy of living cells - New frontiers in membrane and neurobiology. J Neurochem 126, 203–212 (2013).|
|4||C. Eggeling, K.I. Willig, S.J. Sahl, S.W. Hell. Lens-based fluorescence nanoscopy. Q Rev Biophys 48, 178–243 (2015).|
|5||C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova. V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, S.W. Hell. Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457, 1159-1163 (2009).|
|6||C. Eggeling. Super-resolution optical microscopy of lipid plasma membrane dynamics. Essays Biochemistry 57, 69–80 (2015).|