The organization of leukotriene biosynthesis on the nuclear envelope revealed by single molecule localization microscopy and computational analyses.

Angela B Schmider,Hunter L Elliott,Giorgianna E Ellis,Melissa Vaught,Roy J Soberman,Peter A Nigrovic,Nicholas C Bauer,Matthew D Godin,Christianne Bandeira De Melo

doi:10.1371/journal.pone.0211943

Abstract

The initial steps in the synthesis of leukotrienes are the translocation of 5-lipoxygenase (5-LO) to the nuclear envelope and its subsequent association with its scaffold protein 5-lipoxygenase-activating protein (FLAP). A major gap in our understanding of this process is the knowledge of how the organization of 5-LO and FLAP on the nuclear envelope regulates leukotriene synthesis. We combined single molecule localization microscopy with Clus-DoC cluster analysis, and also a novel unbiased cluster analysis to analyze changes in the relationships between 5-LO and FLAP in response to activation of RBL-2H3 cells to generate leukotriene C4. We identified the time-dependent reorganization of both 5-LO and FLAP into higher-order assemblies or clusters in response to cell activation via the IgE receptor. Clus-DoC analysis identified a subset of these clusters with a high degree of interaction between 5-LO and FLAP that specifically correlates with the time course of LTC4 synthesis, strongly suggesting their role in the initiation of leukotriene biosynthesis.

Highlights

All cells must integrate and transduce multiple extracellular signals to achieve an appropriate functional response
Clus-DoC assigns a degree of colocalization (DoC) score to each localization and determines percent colocalization, as well as cluster properties including the number of clusters in a region of interest (ROI), cluster area, and density
Supportive approach, we developed a novel unbiased clustering analysis based on a variable bandwidth mean-shift algorithm for conventional STORM data

Summary

Introduction

All cells must integrate and transduce multiple extracellular signals to achieve an appropriate functional response. The classic pathway of cell activation in response to an allergen is initiated by antigen binding to allergen-specific IgE antibodies coating mast cells via the IgE receptor (FcεR1; FcERI; UniProtKB: P12319) [1,2,3]. Antigen binding triggers the aggregation of FcεR1 receptors, activating their downstream pathways by recruiting a series of kinases to the cytoplasmic tail, and prolongs its presence on the plasma membrane [2, 4,5,6,7]. The kinase cascade sets in motion multiple processes including degranulation and the synthesis of leukotriene B4 (LTB4) [8, 9] and LTC4 [10, 11].

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Results

Conclusion