Follow that cell: leukocyte migration in L-plastin mutant zebrafish

Abstract

Actin dynamics are important in motile cells such as leukocytes which form actin-rich extensions of the plasma membrane. L-plastin (LCP1) is a leukocyte-specific calcium-dependent actin-bundling protein that in mammals is known to affect immune cell migration and synapse formation. Previously, we generated CRISPR/Cas9 engineered zebrafish lacking L-plastin (lcp1-/-) and reported that they had somewhat reduced survival to adulthood, suggesting that lack of L-plastin might negatively affect the immune system. To test this hypothesis, we examined the distribution and migration of neutrophils and macrophages in the transparent tail of early zebrafish larva, using cell-specific markers and an established wound-migration assay. By crossing genotyped parents of our established L-plastin mutant lines, we were able to generate either 1:1 clutches of lcp1 heterozygous: homozygous siblings or 1:2:1 clutches representing all possible sibling combinations of wild type and mutant alleles. Knockout larvae were similar to their heterozygous siblings in having equal body sizes and comparable numbers of neutrophils in caudal hematopoietic tissue at 2 days post-fertilization (dpf), indicating no gross defect in neutrophil production or developmental migration. When stimulated by a tail wound all genotypes of neutrophils were equally migratory in a 2-hour window. However for macrophages we observed both migration defects and morphological differences. L-plastin knockout macrophages still homed to wounds but were slower, less directional and had a star-like morphology with many leading and trailing projections. In contrast, wild type macrophages were faster, more directional, and had a more streamlined, slug-like morphology. Overall, these findings show that in larval zebrafish L-plastin knockout primarily affects the macrophage response with possible consequences for organismal immunity. Consistent with these, we propose a model in which cytoplasmic L-plastin negatively regulates macrophage integrin adhesion by holding these transmembrane heterodimers in a 'clasped', inactive form.