Abstract
ABSTRACTNeutrophils are fast-moving cells essential for host immune functions. Although they primarily rely on glycolysis for ATP, isolated primary human neutrophils depend on mitochondrial membrane potential for chemotaxis. However, it is not known whether mitochondria regulate neutrophil motility in vivo, and the underlying molecular mechanisms remain obscure. Here, we visualized mitochondria in an interconnected network that localizes to the front and rear of migrating neutrophils using a novel transgenic zebrafish line. To disrupt mitochondrial function genetically, we established a gateway system harboring the CRISPR/Cas9 elements for tissue-specific knockout. In a transgenic line, neutrophil-specific disruption of mitochondrial DNA polymerase, polg, significantly reduced the velocity of neutrophil interstitial migration. In addition, inhibiting the mitochondrial electron transport chain or the enzymes that reduce mitochondrial reactive oxygen species also inhibited neutrophil motility. The reduced cell motility that resulted from neutrophil-specific knockout of sod1 was rescued with sod1 mRNA overexpression, or by treating with scavengers of reactive oxygen species. Together, our work has provided the first in vivo evidence that mitochondria regulate neutrophil motility, as well as tools for the functional characterization of mitochondria-related genes in neutrophils and insights into immune deficiency seen in patients with primary mitochondrial disorders.This article has an associated First Person interview with the first author of the paper.
Highlights
Cell motility, crucial for development, immunity, wound healing and cancer metastasis, is central to homeostasis in both health and disease
Mitochondrial dynamics in migrating neutrophils Mitochondria are morphologically dynamic organelles that travel along microtubules (Heggeness et al, 1978), and continuously divide and fuse to form small structures or interconnected networks
We have optimized the original vector developed by the Zon group (Ablain et al, 2015) to allow easier switching of tissue-specific promoters, visualization of Cas9-expressing cells and incorporation of two single-guide RNA (sgRNA)
Summary
Crucial for development, immunity, wound healing and cancer metastasis, is central to homeostasis in both health and disease. Mitochondria are the powerhouse for energy production and integrated regulators for cell signaling, modulating intracellular calcium concentration and apoptosis, to produce metabolic. Disrupting mitochondria membrane potential or prolonged inhibition of the F0-ATPase inhibited neutrophil chemotaxis in 2D, without inducing cell apoptosis or inhibiting the activation of the respiratory burst or phagocytosis (Bao et al, 2015; Fossati et al, 2003). Genetic evidence for the importance of mitochondriarelated pathways in neutrophil chemotaxis is scarce
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