High-Affinity Bent β2-Integrin Molecules in Arresting Neutrophils Face Each Other through Binding to ICAMs In cis

Abstract

SUMMARY Leukocyte adhesion requires β 2 -integrin activation. Resting integrins exist in a bent-closed conformation—i.e., not extended (E − ) and not high affinity (H − )—unable to bind ligand. Fully activated E + H + integrin binds intercellular adhesion molecules (ICAMs) expressed on the opposing cell in trans . E − H − transitions to E + H + through E + H − or through EH + , which binds to ICAMs on the same cell in cis . Spatial patterning of activated integrins is thought to be required for effective arrest, but no high-resolution cell surface localization maps of activated integrins exist. Here, we developed Super-STORM by combining super-resolution microscopy with molecular modeling to precisely localize activated integrin molecules and identify the molecular patterns of activated integrins on primary human neutrophils. At the time of neutrophil arrest, E − H + integrins face each other to form oriented (non-random) nanoclusters. To address the mechanism causing this pattern, we blocked integrin binding to ICAMs in cis , which significantly relieved the face-to-face orientation.

Imaging of the immune system – towards a subcellular and molecular understanding

“…Similar to what was found in neutrophils as described above (Fan et al, 2019), integrin molecules are also important for the migration of T lymphocytes. Interferometric PALM and a constrained photoactivatable fluorescent protein fused to αLβ2 integrin was used to measure the displacement of the head of integrin LFA-1, as a result of its conformational change that is induced on the surface of migrating Jurkat T cells (Moore et al, 2018).…”

“…By using super-resolution imaging, investigators can thus visualize small structures of the cell organelles and cytoskeleton in more detail. Combining STORM with molecular modeling has achieved single-molecule resolution for integrins, including the distribution of molecule nanoclusters, co-localizations and spatial distribution patterns (Fan et al, 2019).…”

“…By using qDF microscopy with conformation-reporting antibodies, we observed an integrin conformation with a bent extracellular domain and a high-affinity headpiece (E − H + ) and its transition to the E + H + conformation on primary live neutrophils (Fan et al, 2016). Furthermore, by combining molecular modeling-based spatial simulation and STORM, we recently developed a technique called SuperSTORM, which allowed us to visualize integrin activation at the singlemolecule level and to identify the number, distribution and arrangement (clustering) of three active conformations (E + H − , E − H + and E + H + ) of β 2 integrins (Fan et al, 2019).…”

“…Lipopolysaccharide (LPS) stimulation increased the size of TLR4 clusters (Aaron et al, 2012). STORM imaging revealed nanoclusters of signal regulatory protein α (SIRPα), Fc gamma receptor I (FcγRI) and Fc gamma receptor II (FcγRII) on human macrophages (Lopes et al, 2017), and β 2 integrins on human neutrophils (Fan et al, 2019). Nanoclusters of FcγRI, but not FcγRII, are constitutively associated with nanoclusters of SIRPα.…”

Frontline Science: A flexible kink in the transmembrane domain impairs β2 integrin extension and cell arrest from rolling

“…3 Thus, KIM127 + mAb24 + β2 integrin molecules are extended and high affinity (E + H + ). 3,12 The E + H + conformation is the only conformation that can mediate arrest from rolling. KIM127 + mAb24 − (E + H − ) β2 integrins mediate transient binding, which results in slow rolling.…”