Super resolution imaging visualized the nanoscaled localization of integrin activation during human neutrophil arrest

Abstract

Adhesion on vascular endothelium is the vital step for leukocyte recruitment and inflammation. Neutrophil is the most abundant leukocyte and is essential for innate immunity and inflammation. The activation of beta2 integrin is critical for neutrophil adhesion. Integrins are bidirectional signaling molecules that are bent at rest. Upon cell activation, integrins can extend (E+) and acquire a high affinity conformation with an “open” headpiece (H+). The canonical “switchblade” model of integrin activation proposes that the E+ conformation precedes H+, and the two are thought to be structurally linked. Only the E+H+ conformation can mediate cell adhesion by binding to ligand in trans. In our previous study, we found out an unexpected bent‐open (E−H+) conformation and an alternative allosteric pathway, which integrin can H+ prior to E+, during human neutrophil arrest. However, the resolution of our previous quantitative dynamic footprinting (qDF) microscopy is limited by optical diffraction limit (~300nm) that we cannot accurately observe the conformation of signal integrin, which is under nanoscale (~20nm). By combining stochastic optical reconstruction microscopy (STORM) with our qDF microscopy, we acquired nanoscale super‐resolution images of integrin activation on arrest human neutrophils. These images confirmed our finding that E−H+ integrin exist and brought us the accurate number and precise local distribution of E−H+, E+H− and E+H+ integrins on arrest neutrophils. We found out that E+H+ integrins are mostly located in microvilli tips and form microscale clusters to support neutrophil arrest.Support or Funding InformationThis research was supported by funding from the National Institutes of Health, USA (NIH, HL078784) and WSA postdoctoral fellowship from the American Heart Association, USA (AHA, 16POST31160014).