Two- and three-color STORM analysis reveals higher-order assembly of leukotriene synthetic complexes on the nuclear envelope of murine neutrophils

J Biol Chem. 2020 Apr 24;295(17):5761-5770. doi: 10.1074/jbc.RA119.012069. Epub 2020 Mar 9.


Over the last several years it has become clear that higher order assemblies on membranes, exemplified by signalosomes, are a paradigm for the regulation of many membrane signaling processes. We have recently combined two-color direct stochastic optical reconstruction microscopy (dSTORM) with the (Clus-DoC) algorithm that combines cluster detection and colocalization analysis to observe the organization of 5-lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) into higher order assemblies on the nuclear envelope of mast cells; these assemblies were linked to leukotriene (LT) C4 production. In this study we investigated whether higher order assemblies of 5-LO and FLAP included cytosolic phospholipase A2 (cPLA2) and were linked to LTB4 production in murine neutrophils. Using two- and three-color dSTORM supported by fluorescence lifetime imaging microscopy we identified higher order assemblies containing 40 molecules (median) (IQR: 23, 87) of 5-LO, and 53 molecules (62, 156) of FLAP monomer. 98 (18, 154) molecules of cPLA2 were clustered with 5-LO, and 77 (33, 114) molecules of cPLA2 were associated with FLAP. These assemblies were tightly linked to LTB4 formation. The activation-dependent close associations of cPLA2, FLAP, and 5-LO in higher order assemblies on the nuclear envelope support a model in which arachidonic acid is generated by cPLA2 in apposition to FLAP, facilitating its transfer to 5-LO to initiate LT synthesis.

Keywords: cell biology; cell signaling; cytokine; direct stochastic optical reconstruction microscopy (dSTORM); leukotriene; neutrophil; nuclear envelope; single-molecule localization microscopy; superresolution microscopy; supramolecular complex.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • 5-Lipoxygenase-Activating Proteins / analysis
  • 5-Lipoxygenase-Activating Proteins / metabolism*
  • Algorithms
  • Animals
  • Arachidonate 5-Lipoxygenase / analysis
  • Arachidonate 5-Lipoxygenase / metabolism*
  • Cell Nucleus / metabolism
  • Cells, Cultured
  • Leukotriene C4 / analysis
  • Leukotriene C4 / metabolism*
  • Mice
  • Mice, Inbred C57BL
  • Microscopy / methods
  • Neutrophils / cytology
  • Neutrophils / metabolism*
  • Optical Imaging / methods


  • 5-Lipoxygenase-Activating Proteins
  • Leukotriene C4
  • Arachidonate 5-Lipoxygenase

Associated data

  • PDB/3O8Y
  • PDB/1CJY
  • PDB/2Q7M