Novel molecular imaging tools for visualising the mechanisms driving vascular-immune cell diapedesis

Overview: A key feature of the body’s adaptive immunity is the ability of immune cells to migrate in and out of the blood circulation. This PhD project will develop and apply a set of state-of-the-art imaging and cell culture tools to visualise the molecular machineries and mechanisms that underpin the close cell-to-cell interactions facilitating this process.

Background: The first step of the migration of leukocytes out of the blood vessels is the attachment to the vascular endothelium in order to gain access to the underlying interstitial space. At the sub-cellular level, this is triggered by cell-to-cell attachment and communication via a range of plasmalemmal receptors [1]. A series of fast intracellular second-messenger signals and rapid remodelling of the membranes and cytoskeletons to form podosomes in both the endothelial cell and leukocyte ensue in a coordinated manner. This mutual motility of the endothelium and leukocytes – a process called ‘diapedesis’ drives the migration of the leukocyte. We will test the hypothesis that the strategic nanoscale organisation of these receptor/ signalling/ cytoskeletal complexes determine the shapes of the podosomes and the direction of diapedesis. Our approach will be to develop and apply a set of organoid and super-resolution imaging tools to gain visual insights into the molecular-scale orchestration of this process. By these molecular patterns and membrane topologies between healthy conditions as well as conditions that mimic local inflammation and local tumours.

Approach: We will establish a three-dimensional (3D) in vitro organoid system of vascular endothelial cells and local chemokine activation of immune cells. To optically map the complex topology and the receptor complexes of the leukocyte-endothelial interface, we will develop a new protocol of a super-resolution microscopy based on the recently-described “enhanced expansion microscopy (EExM)” method [2]. With EExM, we will obtain a 3D molecular-scale fluorescent imprint of the membrane topologies, proteins and other cellular ultrastructures of the endothelial cells and migrating leukocytes onto an acrylamide hydrogel. Hydrating the hydrogel swells and expands this imprint, allowing us to perform super-resolution imaging to build up a spatially-accurate map of the molecules and membrane topologies involved. In order to validate and calibrate the molecular-scale measurements that we will make with EExM imaging, we will also develop a synthetic biomolecular calibration structure which can be embedded into the organoid sample. We will expose the apical and/or basolateral domains of the endothelial cultures to either pro-inflammatory cytokines [3] or tumour chemokines [4] to examine how the plasmalemmal receptor patterns and the podosome structure in disease conditions.

Supervision: The PhD student will be hosted within the laboratory of Dr Izzy Jayasinghe (https://appliedbiophotonics.org/) in The University of Sheffield. They will be given the opportunity to contribute to either existing or new collaborations with academic and/or industrial partners of the group. Co- supervisor of the project will be Dr Barbara Ciani (https://www.sheffield.ac.uk/chemistry/people/academic/barbara-ciani) who will advise on the development of biomolecular calibrants and training in the chemical biology facilities. The student will be a member of the Sheffield-based Imagine-student cohort (http://www.imagine-imaginglife.com/) and will have the opportunity to present novel findings at conferences and public science communication platforms. The PhD training will focus on developing a broad range of cutting-edge research skills including (and not limited to) 3D cell culture, super-resolution microscopy, advanced protein chemistry, advanced image analysis, spatial omics and computer programming. A good understanding of molecular or structural biology and protein chemistry will be desirable, although not essential.