The property of sensing and propagating external cues that drive directional migration is a fundamental property of biological systems, and is essential to physiological and pathological processes including embryogenesis, adult tissue homeostasis, inflammation and immune responses, and metastatic invasion. This proposal aims at understanding how chemotactic signals are packaged and propagated between neighboring cells during chemotaxis. To do so, we study human neutrophils, the most abundant leukocytes in normal human blood. 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 that allows them to efficiently migrate up the fMLF gradient. As they react to fMLF, neutrophils secrete secondary chemoattractants that serve to maintain the robustness and sensitivity to the primary chemoattractant signals. We established that the secondary chemoattractant leukotriene B4 (LTB4) is required for the massive recruitment of neutrophils to sites of injury in vitro and in vivo. In order for LTB4 to act as a bona fide signal relay molecule, it must be released in a form that enables the generation of a stable gradient during chemotaxis. In this context, we established that LTB4 is packaged in vesicles in chemotaxing neutrophils as a way to effectively disseminate gradients between neighboring cells. We found that LTB4 and its synthesizing enzymes ? 5-lipoxigenase (5-LO) and 5-LO activating protein (FLAP) - localize to intracellular multivesicular bodies (MBVs) which, upon chemoattractant stimulation, release their content as exosomes, thereby acting as a packaging mechanism to relay chemotactic signals. Further, we found that MVB biogenesis appears to be initiated at the nuclear envelope (NE) in activated neutrophils. We hypothesize that the NE is a novel site of MVB formation that enables packaging of the LTB4 synthetic machineryinto secretory MVBs that release exosomes to relay of signals during neutrophil chemotaxis. To test this hypothesis, in Aim 1 we will directly visualize 5-LO and FLAP dynamics in live cells using mCherry/GFP fusions and photoactivatable reporters under normal conditions and when endocytosis is blocked. We will also assess the role of FLAP clustering as a driving force for MVB biogenesis at the NE, by generating FLAP mutants with distinct affinities for the 5-LO substrate arachidonic acid. Since integral membrane proteins clustering is considered a hallmark of ordered membrane microdomains, in Aim 2 we will define the role of nuclear lipid micro-domains in MVB biogenesis. Finally, in Aim 3 we will establish the role of membrane remodeling complexes in the formation of the nuclear MVBs by assessing the role of ESCRTs in this process and identify accessory proteins involved in NE remodeling. This project is poised to provide much needed insight into the mechanisms regulating the genesis of chemotactic signals during neutrophil chemotaxis and will bring unprecedented knowledge into the role of the NE in the biogenesis of MVBs and in the interplay between lipid- and ESCRT-dependent pathways in their biogenesis.
The rapid recruitment of white blood cells to sites of tissue injury or infection is a vital determinant of health and its deregulation can lead to chronic disease. This proposal aims at understanding how chemotactic signals are packaged and propagated between neighboring neutrophils, a type of white blood cell critical to innate immunity responses, as they are being recruited to sites of injury or infection. We propose to identify the mechanisms by which exosomes, which are small membrane-bound elements with a key role in signal propagation, are being generated intracellularly from multivesicular bodies using cell biological, biochemical and genetic approaches.
