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

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.

Fatty acid binding protein 5 forms higher order assemblies with FLAP or COX-2 in LPS-stimulated macrophages

“…Multiple changes in the relationship of FABP5:FLAP were identified (Figure 5) after stimulation with LPS. Because we previously observed strong clustered interactions of FLAP, 5-LO, and cPLA 2 associated with the synthesis of LTs (18, 19) we focused on the changes we observed in HICs with FABP5. The largest increases between FABP5:FLAP following LPS exposure were in the cluster area of HIC, and the relative density of FLAP clusters ( Figure 5C and E ).…”

“…Two-color dSTORM experiments were performed using a Nikon system previously described (18, 19) Approximately 300,000 cells per well were added to an 8-well chamber slide (Ibidi) and allowed to adhere for 24 hours in normal incubating conditions. The cells were then stimulated with 100 ng/mL LPS for 18 hours (as described above) or left untreated.…”

“…Primary antibodies, anti-FABP5 (Thermo Fisher, 1:100), anti-FLAP (Novus Biologicals, 1:100), anti-COX-1 (Invitrogen, 1:100) and anti-COX-2 (Santa Cruz, 1:100) were diluted in blocking solution overnight at 4 °C. Cells were then washed twice in PBS and incubated with in-house conjugated secondary antibodies (3 μg/mL) for 60 min at RT in the dark (18, 19). For two-color dSTORM experiments, donkey anti-rabbit Alexa Fluor 647, donkey anti-goat Alexa Fluor 488 and donkey anti-mouse Atto 488 were used.…”

“…As done previously in our laboratory (18, 19) we employed Clus-DoC (32) to quantify colocalization of individual proteins and molecules (localizations) and cluster properties in regions of interest (ROI). ROIs were drawn around the nuclear envelope and endoplasmic reticulum.…”

“…All experiments were performed at room temperature on a Nikon system equipped with Becker and Hickl DCS-120 TCSPC system (18, 19). Cells were added to an 8-well chamber slide (Millipore) and allowed to adhere for 24 hours in normal incubating conditions.…”