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3D Super-Resolution Correlative Light-Electron Microscopy of the Protein Complexes


Project Description

Inflammation plays an important role in the development and progression of cardiovascular diseases. The modulation of inflammation and immunity by CD4+ regulatory T-cells has therefore received increasing attention. [1] The development of advanced and single-molecule sensitive fluorescence microscopy methods now enables quantitative assessment of involved protein complexes at the ultrastructural level to further improve existing models of immune cell signalling. [2] To gain a better understanding of the morphology and composition of immunological synapses, we will study the distribution of key proteins using correlative light-electron microscopy (CLEM) to discriminate between vesicular and plasma-membranous pools. [3] Furthermore, the development of quantitative approaches for 3D CLEM will enable additional collaborations within COMPARE (Centre of Membrane Proteins and Receptors).

The COMPARE seminars offer the successful applicant to learn about potential collaborations in the field of membrane protein/receptor clustering and to present the progress of the PhD project on a regular basis. The project is linked additional COMPARE studentships working on advanced methods for molecular counting.

Project outline:

Year 1: Establishing an automated microscopy platform for high-throughput single-molecule super-resolution microscopy

In the first year, the student will extend a previously established epifluorescence microscope for fully automated single-molecule microscopy. This will include implementation of several electronic and optical devices using commercially available and custom-built components. All hardware will be integrated into an available microscope control software based on the MicroManager open-source project. This part of the project will be supported by postdocs in the lab with prior experience in microscope design. With the established setup, a standardized imaging workflow for fully autonomous imaging will be developed. The student will furthermore optimize sample preparation protocols for correlative light and electron microscopy based on approaches previously established in the lab. In collaboration with Iain Styles (Computational Sciences) the data analysis for alignment and correlation of the multidimensional image stacks will be approached.

Year 2: Quantitative microscopy

The results from year 1 will be complemented with additional experiments to extract quantitative information about protein abundances. For this, different methods for molecular counting have previously been established in the lab and will be transferred to a CLEM workflow in the scope of this project. This includes bleach-step analysis as well as our own approach “counting by photon statistics” (CoPS) as well as calibration protocols for quantification of the labelling efficiencies in cells.

Year 3: 3D super-resolution correlative light-electron microscopy in T-cells

The LAT-signalosome in T-cells will be analyzed in a quantitative 3D CLEM approach. Of particular interest within this project will be the distribution of the LAT pool in T-cells before and after immune activation. Vesicular (intracellular) and plasma membrane-bound LAT will be identified by combining the resolution of electron microscopy with molecule-specific fluorescence microscopy. Several stable cell lines for fluorescent labeling of adapter proteins such as LAT and SLP76 with protein tags have already been established in the lab and will be used to study micro cluster formation and reorganization during T-cell activation. [5-7] The studies will be extended to other protein complexes, such as platelet immunoglobulin C-type lectin-like receptors GPVI and CLEC-2 (with Steve Watson, Steve Thomas and Natalie Poulter, ICVS) which are novel targets for a variety of thrombosis and thrombo-inflammatory disorders.

Funding Notes

This is a 3-year PhD fellowship funded by COMPARE (The Centre of Membrane Proteins and Receptors). Interested students should contact Prof Herten (). Please send a covering letter highlighting your research experience and interests and also include a detailed CV (including nationality and country of birth), names and addresses of two referees, copies of your degree certificates with transcripts, evidence of your proficiency in the English language, if applicable.

References

[1] Meng et al. Nat. Rev. Cardiol. 13, 167 (2016), doi: 10.1038/nrcardio.2015.169.
[2] Grussmayer et al. Meth. Appl. Fluoresc. 7, 012003 (2019), doi: 10.1088/2050-6120/aaf2eb.
[3] Gaietta et al., Science 296, 503 (2002), doi: 10.1126/science.1068793.
[4] Grussmayer et al. PCCP 19, 8962 (2017), doi: 10.1039/C7CP00363C.
[5] Abraham et al. J. Immunol. 189, 1898 (2012), doi: 10.4049/jimmunol.1200652.
[6] Pan et al. Cell Microbiol. 15, 1605 (2013), doi: 10.1111/cmi.12148.
[7] Abraham et al. Cell Commun. Signal. 10, 39 (2012), doi: 10.1186/1478-811X-10-39.

More information:

The Centre of Membrane Proteins and Receptors (COMPARE): http://www.birmingham-nottingham.ac.uk/compare/

The Institute of Cardiovascular Sciences: https://www.birmingham.ac.uk/research/activity/cardiovascular-sciences/index.aspx

The School of Chemistry: https://www.birmingham.ac.uk/schools/chemistry/index.aspx

Postgraduate studies at the University of Birmingham: https://www.birmingham.ac.uk/postgraduate/prospectus/index.aspx

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