Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Tuesday Speaker Abstracts

THE NEUTROPHIL NUCLEUS AS A SIGNALING HUB DURING NEUTROPHIL CHEMOTAXIS Subhash B Arya 1 ; Song Chen 1,2 ; Fatima Jordan-Javed 1,3 ; Shuvasree SenGupta 1 ; Carole A. Parent 1,2,3 ; 1 University of Michigan, Life Sciences Institute, Ann Arbor, MI, USA 2 University of Michigan, Department of Pharmacology, Ann Arbor, MI, USA 3 University of Michigan, Department of Cell and Developmental Biology, Ann Arbor, MI, USA When exposed to primary chemoattractants like N-formyl-Met-Leu-Phe (fMLF), which is secreted by pathogens invading the body and by necrotic cells at sites of injury, neutrophils rapidly undergo polarization and migrate up the fMLF gradient. In response to fMLF, neutrophils secrete the secondary chemoattractant leukotriene B 4 (LTB 4 ), which dramatically amplifies the recruitment range of neutrophils to sites of infection or injury. LTB 4 is synthesized from arachidonic acid through the action of the cytosolic enzyme 5-lipoxygenase (5-LO), the endoplasmic reticulum/nuclear envelope-resident protein 5-lipoxygenase activating protein (FLAP) and leukotriene A 4 hydrolase. Interestingly, upon fMLF activation, we discovered that LTB 4 synthesizing enzymes and LTB 4 are packaged in and released from extracellular vesicles called exosomes. Additionally, we determined that the biogenesis of LTB 4 -containing exosomes originates at the nuclear envelope of activated neutrophils. We found that the neutral Sphingomyelinase 1 (nSMase1)-dependent generation of ceramide-rich lipid-ordered membrane microdomains is required for FLAP and 5-LO clustering at the NE, and subsequent nuclear envelope-budding. We also found that ALIX, CHMP4B and CHMP7 localize inside nuclear envelope-derived buds and cytosolic vesicles in migrating neutrophils. Using nano-scale microscopic resolution achieved by 4x isotopic expansion of samples, we found 5-LO-positive intraluminal vesicles within nuclear envelope-resident Lamin B Receptor-positive limiting membranes, generating nuclear envelope-derived multivesicular bodies (NE-MVBs). Interestingly, we discovered that both NE-MVBs and exosomes are distinct from conventional plasma membrane-derived CD63-positive MVB/exosomes. Together, these findings elucidate the mechanism by which LTB 4 is released from neutrophils and identify a non-conventional pathway for exosome generation.

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