Neutrophils migrate rapidly towards a site of inflammation and mediate bacterial killing
through highly regulated pathways that involve the phagocytosis of bacteria and the
generation of reactive oxygen species by the NADPH oxidase complex. The Rac small
GTPases have prominent roles in the regulation of neutrophil signaling pathways but the
research strategies used to analyze their functions in live cells have been limited, since
neutrophils are terminally differentiated and difficult to manipulate genetically. In this
thesis, I describe a novel high efficiency protocol for transiently transfecting neutrophils
that allowed me to investigate the roles of Rac1 and Rac2 in neutrophils in a completely
new way, in real time. Using this technique, I show that a bacterial protein known to
inhibit chemotaxis in vitro, selectively inhibits Rac1 activation downstream of fMLP
stimulation and inhibits neutrophils polarization. Further dissecting the roles of Rac
isoforms, I used various approaches to show that Rac1 and Rac2 differentially regulate
free-barbed end (FBE) formation downstream of the fMLP receptor. Rac1 is responsible
for ~30% of FBE whereas Rac2 is the regulator of FBE formation (~70%) through the
activation of cofilin and Arp2/3. Finally, these observations led to the analysis of the
mechanisms underlying the Rac1 and Rac2 functions. I show that membrane charge
determines Rac1 and Rac2 differential localization during phagocytosis and chemotaxis
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based on their different aminoacid residues in the polybasic domain. This mechanism
depends on lipid metabolism and the accumulation of negatively charged lipids at cellular
membranes. During chemotaxis, neutrophils have a polarized accumulation of negatively
charged lipids at the leading edge membrane that selectively recruit Rac1. In contrast, the
lipid metabolism that occurs at the phagosome membrane decreases its negativity and
selectively recruits Rac2. All together, this thesis describes the study of primary
neutrophil functions from a new angle and adds some valuable information to the
comprehension of effective neutrophil activation based on the analysis of Rac isoforms.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/17798 |
| Date | 24 September 2009 |
| Creators | Magalhaes, Marco Antonio de Oliveira |
| Contributors | Ellen, Richard P., Glogauer, Michael |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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