Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Poster Abstracts

53-POS Board 18 THE ROLE OF FLAP IN LTB4 SYNTHESIS DURING SIGNAL RELAY IN NEUTROPHILS

Samuel P Collie 1 ; Subhash B Arya 3 ; Carole A Parent 1,2,3 ; 1 University of Michigan, Cellular and Molecular Biology, Ann Arbor, MI, USA 2 University of Michigan, Department of Pharmacology, Ann Arbor, MI, USA 3 University of Michigan, Life Sciences Institute, Ann Arbor, MI, USA

Neutrophils are first responders to sites of injury and infection and play essential roles as mediators of inflammation. They reach injured and infected sites by recognizing gradients of chemical cues such as the peptide N-Formylmethionyl-leucyl-phenylalanine (fMLF) and migrating directionally towards them. fMLF activation also leads to the secretion of the secondary chemoattractant Leukotriene B 4 (LTB 4 ), which amplifies fMLF-mediated responses and increases the recruitment range of neutrophils. Interestingly, LTB 4 synthesis is initiated at the nuclear envelope of migrating neutrophils through the action of the 5-Lipoxygenase (5-LO), 5 LO Activating Protein (FLAP), and Leukotriene A 4 Hydrolase from the substrate arachidonic acid (AA). Upon fMLF activation, these LTB 4 synthesizing enzymes are packaged in nuclear envelope-derived buds that go on to form multivesicular bodies (MVBs), which fuse with the plasma membrane and release their intraluminal vesicles as exosomes that release LTB 4 to the extracellular environment. The mechanisms that lead to the formation of nuclear envelope buds are poorly understood. We found that FLAP, a transmembrane protein, is homogenously distributed on nuclear envelope buds, where it co-occurs with ceramide in an nSMase-dependent fashion. Furthermore, FLAP is enriched in MVBs and LTB 4 -containing exosomes. As AA binding has been proposed to be required for FLAP trimerization and FLAP/5-LO interaction, we hypothesize that AA-induced FLAP trimerization facilitates the molecular crowding necessary for the initiation of nuclear envelope budding. We also envision that FLAP is acting as a scaffold to recruit proteins that promote the formation of nuclear buds, such as ESCRT. We are currently generating FLAP mutants defective in AA binding, testing pharmacological inhibition of FLAP in migrating neutrophils and working on FLAP pull-down assays coupled to mass spectrometry to test our hypotheses.

90