The role of nuclear dynamics in migrating neutrophils during immune responses in vivo
Project ID: II12
Supervisor: Dr. MILKA SARRIS
Cell movement is essential for development and integrity of animal organisms and of special importance in immune responses. Leukocytes are professional migratory cells that routinely patrol the body for signs of infection or damage and accurately navigate through virtually any body tissue. Migration of these cells is generally confronted with physical confinement not only when extravasating across venular endothelium but also when moving through narrow tissue interstices. To this end, leukocytes use flexible changes in cell shape that allow squeezing through tight structures. These deformations pose a challenge to the cell nucleus and leukocytes have evolved specific nucleoskeletal architectures to endow minimal rigidity to this compatment. Nevertheless, recent studies show that during movement in confinement in vitro the integrity of the nucleus is often breached leading to leakage of cytoplasmic components. The ESCRT III complex, a protein complex able to remodel membranes, is recruited to the nucleus and restores nuclear envelope integrity, limiting DNA damage and cell death. These in vitro studies raise the question to what extend leukocytes are affected by nuclear disruption during migration in vivo and how this functionally influences immune responses. To address this, we will use live zebrafish embryos , which are transparent and thus ideal for monitoring leukocyte migration behaviour and dynamics . We will use specific fluorescent markers to label nuclear and cytoplasmic components and track nuclear rupture events in vivo. Using a transgenic line with photoconvertible neutrophils we will track the fate of neutrophils undergoing nuclear rupture. We will assess the role of the ESCRT III repair complex in maintaining nuclear integrity of migrating leukocytes, through a new conditional, tissue-specififc CRISPR/Cas9 technology that we have developed. We will use bacterial infection models in zebrafish to establish the physiological relevance of nuclear rupture and repair in the survival of leukocytes and their ability to clear infection.
1. Kolaczkowska, E. & Kubes, P. Neutrophil recruitment and function in health and inflammation. Nat Rev Immunol 13, 159–175 (2013).
2. Raab et al. ESCRT III repairs nuclear envelope ruptures during cell migration to limit DNA damage and cell death. Science 15, 352-62 (2016).
Once we have identified the physiological role of nuclear envelope repair through studies in zebrafish, we will translate our findings to human cells, in collaboration with the group of Edwin Chilvers. This can be done by transplanting labelled human neutrophils into zebrafish where they can be subsequently followed in real tissue settings. We will test chemical inhibitors that may be able to affect the process of nuclear repair.