Lattice Light Sheet Imaging of Neutrophil Cytoplasmic and Nuclear Plasticity in Vivo

The peripatetic capability of neutrophils underpins their function in tissue maintenance/repair and in antimicrobial defence. An extraordinary degree of cellular plasticity has long been regarded as essential for their migratory capacity. Until now, it has been challenging to document the instantaneous morphology of individual migrating neutrophils in vivo because of their random distribution through tissues, their rapid speed, and the extreme moment-to-moment dynamic complexity of their form as they explore and traverse the 3-dimensional space of living tissues. To assess the relationship between neutrophil plasticity and their migratory activity, we have examined the morphology of stationary and migratory neutrophils in vivo in unprecedented 4-dimensional detail. To achieve this, we combined the optical transparency of zebrafish embryos, specific reporter transgenes, and the technical advantages of lattice light sheet microscopy (LLSM) for high subcellular spatiotemporal resolution with minimal phototoxicity. To enable concurrent assessment of cytoplasmic and nuclear form, we selectively labelled the neutrophil cytoplasmic membrane with mCherry-Caax and the nuclear envelope with EGFP-Lamin transgenes. Descriptive and quantitative morphometric approaches are used to correlate nuclear shape, arborisation, distortion and position with cytoplasmic form and migratory status. Analysis of numerous individual stationary and migrating wild-type neutrophils has revealed principles behind the complex dynamic relationship between cytoplasmic and nuclear forms and mobility status. Furthermore, we specifically hypothesize that nuclear plasticity and biomechanics influence neutrophil migratory behaviours. To test this by functionally altering nuclear biomechanics independently of cytoplasmic structure, we have made zebrafish mutants deficient in structural nuclear proteins (Lamins A/C, B1, B2, and Lamin-B receptor), and made genetic tools to overexpress them. Collectively, these studies have already revealed a substantially refined picture of neutrophil morphology in vivo, and provide a basis for exploring the relationship between nuclear plasticity and neutrophil function.