Effective Neutrophil Activation During Innate Immunity: Understanding the Specific Roles of Rac1 and Rac2
Year: 2009
Type: dissertation
Abstract: Neutrophils migrate rapidly towards a site of inflammation and mediate bacterial killing\nthrough highly regulated pathways that involve the phagocytosis of bacteria and the\ngeneration of reactive oxygen species by the NADPH oxidase complex. The Rac small\nGTPases have prominent roles in the regulation of neutrophil signaling pathways but the\nresearch strategies used to analyze their functions in live cells have been limited, since\nneutrophils are terminally differentiated and difficult to manipulate genetically. In this\nthesis, I describe a novel high efficiency protocol for transiently transfecting neutrophils\nthat allowed me to investigate the roles of Rac1 and Rac2 in neutrophils in a completely\nnew way, in real time. Using this technique, I show that a bacterial protein known to\ninhibit chemotaxis in vitro, selectively inhibits Rac1 activation downstream of fMLP\nstimulation and inhibits neutrophils polarization. Further dissecting the roles of Rac\nisoforms, I used various approaches to show that Rac1 and Rac2 differentially regulate\nfree-barbed end (FBE) formation downstream of the fMLP receptor. Rac1 is responsible\nfor ~30% of FBE whereas Rac2 is the regulator of FBE formation (~70%) through the\nactivation of cofilin and Arp2/3. Finally, these observations led to the analysis of the\nmechanisms underlying the Rac1 and Rac2 functions. I show that membrane charge\ndetermines Rac1 and Rac2 differential localization during phagocytosis and chemotaxis\niii\nbased on their different aminoacid residues in the polybasic domain. This mechanism\ndepends on lipid metabolism and the accumulation of negatively charged lipids at cellular\nmembranes. During chemotaxis, neutrophils have a polarized accumulation of negatively\ncharged lipids at the leading edge membrane that selectively recruit Rac1. In contrast, the\nlipid metabolism that occurs at the phagosome membrane decreases its negativity and\nselectively recruits Rac2. All together, this thesis describes the study of primary\nneutrophil functions from a new angle and adds some valuable information to the\ncomprehension of effective neutrophil activation based on the analysis of Rac isoforms.
Source: TSpace (University of Toronto)
Author: Magalhaes MAdO
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Award: G839646784
OpenAlex ID: W2486277627