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Squeezing through small gaps: Understanding nuclear mechanics during invasive migration

Primary Supervisor: Dr Aparna Ratheesh, Warwick Medical School

Secondary supervisor: Dr Anne Straube

PhD project title: Squeezing through small gaps: Understanding nuclear mechanics during invasive migration

University of Registration: University of Warwick

Project outline:

Background: Macrophages are highly migratory immune cells capable of engulfing and removing dead cells, debris and pathogens. In adults, tissue resident macrophages (TRMs), a subset of macrophages, serve important functions during homeostasis and drive pathological conditions including vascular and neurodegenerative diseases and cancer. In the last decade, several studies showed that embryonic migration followed by tissue infiltration sets up macrophage populations which persist into adults and disproved the conventional paradigm that TRMs are derived solely from circulating adult monocytes1. However, there is a huge gap in our understanding of the biochemical and biophysical mechanisms and kinetics of how embryonic macrophages invade tissues in whole organisms and my lab uses the embryonic migration of the fruit fly Drosophila Melanogaster macrophages as a model system to understand this process better. We have shown that embryonic macrophage migration requires them to squeeze (average diameter 10-15 microns when unconfined) into a tissue interface with no pre-existing gaps2. Changes in nuclear mechanics is an essential determinant of confined migration downstream of cytoskeletal alterations3. In this project, we will dissect how nuclear mechanics is regulated by the extrinsic environment and define the force generating mechanisms within the macrophages which deform the nucleus. Finally, we will explore how changes in nuclear shape can trigger changes in gene expression and macrophage function.

Objectives:

  1. How does the nucleus deform? Determine the intrinsic and extrinsic molecular and mechanical regulators of nuclear deformation under confinement during migration.
  2. How does nuclear deformation affect macrophage migration and function?

Methods: Drosophila genetics, optogenetics, live imaging of embryos using a confocal and two photon microscope, FACS sorting, ex vivo migration and confinement assays, biophysical manipulations, quantitative image analysis and mathematical modelling.

References:

  1. Stremmel C et al. Yolk sac macrophage progenitors traffic to the embryo during defined stages of development. Nature Communicationsvolume 9, Article number: 75 (2018)
  1. Ratheesh A et al. Drosophila TNF modulates tissue tension in the embryo to facilitate macrophage invasive migration. Developmental Cell. 2018 May 7;45(3).
  2. Thiam H et al. Perinuclear Arp2/3-driven actin polymerization enables nuclear deformation to facilitate cell migration through complex environments. Nature Communications 2016.

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

    Techniques that will be undertaken during the project:

    • Drosophila genetics
    • Optogenetics
    • RNA interference technology and CRISPR/Cas9
    • Two-photon and Dual Inverted Selective Plane Illumination Microscopy (diSPIM)
    • Laser Ablation
    • Cell culture techniques
    • Migration and confinement assays
    • FACS sorting
    • Quantitative image analysis

    Contact: Dr Aparna Ratheesh, University of Warwick