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Mechanical and biochemical regulation of macrophage migration by TNF receptors

Principal Supervisor: Dr Aparna Ratheesh, Warwick Medical School

Secondary supervisor: Dr John James

PhD project title: Mechanical and biochemical regulation of macrophage migration by TNF receptors

University of Registration: University of Warwick

Project outline:

Current Knowledge

The ability of cells to cross tissue barriers is a crucial parameter governing several physiological and pathological events. Vertebrate immune cells cross blood vessels in order to reach sites of infection (1). Metastatic cells similarly traverse blood vessels to spread throughout the body (2). These processes require coordinated biochemical and biophysical alterations in both the invading cells and the surrounding tissues. While this has been studied much in vitro, not much is known about how this happens within a 3-dimensional environment in vivo.

To dissect the alterations accompanying invasion and underlying mechanisms, we study the invasive migration of macrophages into a tissue referred to as “germband” during Drosophila embryogenesis. Invasion occurs specifically at the interfaces of tissues with differing cortical tension in the germband. We have established a crucial role for the Drosophila Tumor Necrosis Factor (TNF), Eiger, in mediating this process. Eiger, which is released from surrounding tissues, acts non-autonomously through one of its receptors to lower the cortical tension of ectoderm in the germband allowing the macrophages to deform ectoderm which facilitates invasion (3). Interestingly, our data showed that removing Eiger only in the macrophages through genetic means, showed a decrease in invasion, suggesting a potential role for the cytokine within macrophages themselves which is so far unexplored. In this project, we would like to investigate the role of Eiger signalling within macrophages during their migratory process. Our preliminary data shows a role for Eiger receptors in modulating macrophage migration through modifications of Actin cytoskeleton. We hypothesize that Eiger receptors modify macrophage Actin cytoskeleton to alter cellular mechanics, protrusion formation and nuclear deformations to regulate macrophage migration.

Experimental Methods

This is an interdisciplinary project and will involve Drosophila genetics, live imaging of Drosophila embryos as well as ex vivo macrophages, biophysical experiments and sophisticated image analysis and quantification. The student will learn handling of Drosophila adult flies utilize the Gal4-UAS system in Drosophila to interfere genetically with the Eiger receptors and their downstream partners. We have transgenic lines in house, which expresses both Gal4 under the macrophage promoter and fluorescent tags directly coupled to the macrophage promoter allowing simultaneous manipulation and visualization of macrophages. Quantitative live imaging using two-photon and confocal microscopy will be used to assess macrophage migration as well as the receptor dynamics and localization. We have preliminary evidence that Eiger signalling affects the cytoskeleton and ex vivo macrophages will be used to identify cytoskeletal interactors of TNF signalling and genetic and biochemical interaction assays will be used to dissect the signalling pathway in macrophages. Ex vivo migration assays will be used to assess the role of Eiger receptors in modifying Actin flows and Actomyosin contractility. Finally, we will test whether cortical tension and force generation in macrophages is regulated by this signalling pathway.

References:

  1. Muller, W.A., 2011. Mechanism of leukocyte transendothelial migration. Annu Rev Pathol, 6, pp.323–344.
  2. Hanahan, D. et al., 2011. Hallmarks of Cancer: The Next Generation. Cell, 144(5), pp.646–674.
  3. Ratheesh A et al. Drosophila TNF modulates tissue tension in the embryo to facilitate macrophage invasive migration. Developmental Cell. 2018 May 7;45(3).

BBSRC Strategic Research Priority: Understanding the rules of life: Immunology

    Techniques that will be undertaken during the project:

    • Drosophila genetics
    • RNA interference technology and CRISPR/Cas9
    • Two-photon and Dual Inverted Selective Plane Illumination Microscopy (diSPIM)
    • Laser Ablation
    • Gel deformation assays

    Contact: Dr Aparna Ratheesh, University of Warwick