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Multi-modal imaging of the organisation of the cytoskeleton in platelet thrombi

Primary Supervisor: Dr Steve Thomas, Institute of Cardiovascular Sciences

Secondary supervisor: Dr Natalie Poulter

PhD project title: Multi-modal imaging of the organisation of the cytoskeleton in platelet thrombi

University of Registration: University of Birmingham

Project outline:

The project will combine expansion, light sheet and super-resolution microscopy to understand how the cytoskeleton and cell-cell junctions contribute to thrombus formation.

Thrombus formation is a fundamental process by which animals are able to respond to injury to prevent blood loss and to promote wound repair. In mammals, platelets are the blood cells that play a critical role this process. The formation of a thrombus is a complex process that involves dynamic interactions between endothelial cells and sub-endothelial matrix proteins of the vessel wall, circulating platelets and the coagulation cascade. It occurs in 3D over time and is influenced by a range of receptors, signalling molecules, plasma components and blood flow. Recent work using intra-vital microscopy has established that individual thrombi are heterogeneous structures which consist of various regions including an inner core of P-selectin positive fully activated platelets which support fibrin formation and an outer shell of loosely adherent platelets which are either resting or only partially activated1. This heterogeneity reflects the spatial and temporal nature of platelet activation during thrombus formation and requires coordination of signalling events and cytoskeletal organisation. Published work has identified the existence of cell-cell interactions (Gap junctions) between platelets which facilitate intercellular communication2. Our recent preliminary data suggests that the cytoskeleton in platelet rich thrombi is continuous between adjacent platelets and may therefore contribute to the formation, stabilisation and turnover of thrombi. To understand the cytoskeletal processes at play during thrombus growth and regulation, we need to visualise them and to identify the junctional complexes involved in this organisation. However, this is technically challenging as platelet aggregates are large, complex structures and not amenable to traditional fluorescence microscopy approaches. To address this challenge we are applying a combination of advanced microscopy approaches to enable us to visualise platelets and sub-cellular structures within thrombi.

Expansion microscopy is a unique approach to generating super-resolution images of complex, large samples3. It involves embedding samples within a polymer, linking proteins of interest to this gel and then expanding the sample to between 4 and 10 times its original volume. This simultaneously provides super-resolution images and clears the sample at the same time, meaning it can provide detailed images of previously intractable samples. By imaging these expanded samples using light sheet fluorescence microscopy (LSFM) (ref) we can obtain 3D volumes of platelet thrombi and determine the localisation and organisation of the cytoskeleton and junctional complexes within them. Therefore, the aim of this project is to apply Expansion and LSFM to study the role of the cytoskeleton and cell-cell junctions in thrombus formation, in order to increase our understanding of this fundamental mammalian response to blood vessel damage.

The student will be based in the Birmingham Platelet Group at the University of Birmingham medical school and will learn cellular and molecular techniques for studying platelet biology as well as using state-of-the-art imaging technology to address fundamental biological questions regarding platelet activation and thrombus formation. We will use and develop LSFM to image these events and ask questions such as “Which type of cell-cell junctions occur in thrombi?” and “how is thrombus formation affected by agents that block the cytoskeleton or junction formation?” This work will advance our understanding of the biology and dynamic nature of this fundamental mammalian process.

References:

  1. Ivancui & Stalker, 2015, JTH doi: 10.1111/jth.13145
  2. Vaiyapuri et al, 2015, Biochem Soc Trans. 43(3):489-943.
  3. Faulkner et al, 2020, Int J biochem, Cell Biol. doi: 10.1016/j.biocel.2020.105764

BBSRC Strategic Research Priority: Understanding the Rules of Life: Immunology

Techniques that will be undertaken during the project:

  • Platelet preparation from both mouse and humans
  • Platelet function assays
  • In vitro flow adhesion assays
  • Advanced fluorescence microscopy including Light Sheet Microscopy, Expansion Microscopy and Super-Resolution Microscopy
  • Development of protocols for appropriate sample presentation for expansion microscopy and light sheet imaging.
  • Computational image analysis

Contact: Dr Steve Thomas, University of Birmingham