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

Abstract

1The initial steps in the synthesis of leukotrienes are the translocation of 5-lipoxygenase 2 (5-LO) to the nuclear envelope and its subsequent association with its scaffold protein 5-3 lipoxygenase-activating protein (FLAP). A major gap in our understanding of this process is the 4 knowledge of how the organization of 5-LO and FLAP on the nuclear envelope regulates 5 leukotriene synthesis. We combined single molecule localization microscopy with Clus-DoC 6 cluster analysis, and also a novel unbiased cluster analysis to analyze changes in the 7 relationships between 5-LO and FLAP in response to activation of RBL-2H3 cells to generate 8 leukotriene C 4 . We identified the time-dependent reorganization of both 5-LO and FLAP into 9 higher-order assemblies or clusters in response to cell activation via the IgE receptor. Clus-DoC 10 analysis identified a subset of these clusters with a high degree of interaction between 5-LO and 11 FLAP that specifically correlates with the time course of LTC 4 synthesis, strongly suggesting 12 their role in the initiation of leukotriene biosynthesis. 13One way cells integrate signals is by the organization and disassembly of higher order 26 multiprotein assemblies; especially those regulated by transient weak interactions [13, 14]. 27These structures must be assembled at the right time with the correct spatial localization. 28Modulating their composition and organization by orchestrating changes in the relationships 29 between member proteins, such as by post-translational modification, can determine cellular 30 responses to stimuli [13][14][15]. 31 Leukotrienes (LTs), arachidonate 5-lipoxygenase (5-LO; UniProtKB: P09917) products 32 of arachidonic acid (AA) metabolism, play a major role in initiating and amplifying inflammatory 33 diseases, ranging from asthma to cardiovascular disease. Because of the dire consequences of 34 the unregulated activation of this pathway, cells have evolved a series of complex control 35 mechanisms to prevent the inadvertent initiation of LT synthesis. One strategy is based on the 36 spatial segregation of the biosynthetic enzymes in different cellular compartments. In resting 37 cells, 5-LO is localized in the nucleus and cytosol, and cytosolic phospholipase A 2 (cPLA 2 ; 38 UniProtKB: P47712) is in the cytosol [16][17][18][19][20][21]. In activated mast cells, calcium influx triggers 39 translocation of cPLA 2 to the Golgi and ER membranes [16, 17], and of 5-LO to the nuclear 40 envelope [19, 21], a main site LT synthesis. cPLA 2 releases AA from the membrane 41 phospholipids. To form the core of the LT synthetic complex on the nuclear envelope, [22, 23], 42 AA associates with the homotrimeric integral membrane scaffold protein, arachidonate 5-43 lipoxygenase-activating protein (FLAP; UniProtKB: P20292). This event alters the relationship 44 between the N-and C-terminal domains of FLAP [23] leading to recruitment of membrane-45 associated 5-LO [22, 23]. Though this is a transient, weak interaction requiring chemical 46 crosslinking to identify biochem...

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

“…Neutrophil stimulation was conducted in chambered cover glass slides, so that we could directly associate LTB4 levels with the molecular organization of 5-LO, cPLA2 and FLAP in the same cells. We imaged the cells with two-color dSTORM, employing the workflow demonstrated in our recent publication where dSTORM localization data was converted to .txt files then analyzed by Clus-DoC in MATLAB (40).…”

“…Coclusters can be further divided into subsets based on parameters such as the degree of interaction. In mast cells, Clus-DoC allowed us to link the properties of clusters of 5-LO and FLAP to the generation of LTC4 (40). We applied the same algorithm to determine cluster properties in neutrophils.…”

“…We have previously combined two-color dSTORM analysis with the Clus-DoC cluster algorithm to identify higher order assemblies of 5-LO and FLAP on the nuclear envelope that are linked to LTC4 formation in the RBL-2H3 mast cell line (40). These assemblies averaged 20 molecules of 5-LO and the same number of FLAP molecules.…”

Arachidonic acid promotes the binding of 5-lipoxygenase on nanodiscs containing 5-lipoxygenase activating protein in the absence of calcium-ions

“…It would be interesting to see if these would become structured under other conditions e.g. close to other proteins (10,17,38) or would remain disordered. Simply the location of FLAP in a lipid environment could have induced order, however, specific protrusions from organised C-terminal FLAP extensions in the non-complexed FND:s could not be observed at our resolution in negative stain (Fig.…”

A cross–nearest neighbor/Monte Carlo algorithm for single-molecule localization microscopy defines interactions between p53, Mdm2, and MEG3

“…Cells were washed with DPBS 6 times for 5 min each. Secondary antibodies (donkey anti-rabbit IgG (Jackson ImmunoResearch) and donkey anti-mouse IgG (Jackson ImmunoResearch)) were labeled as previously described with ATTO 488 (ThermoFisher Scientific) or Alexa Fluor 647 (ThermoFisher Scientific) for a dye ratio of ~1:1 (Schmider et al, 2019). Secondary antibodies were added at 3 μg/mL each in blocking buffer and incubated for 2 h at room temperature in the dark.…”