Title

Regulation of chemotaxis in mouse neutrophils

Date of Completion

January 2007

Keywords

Biology, Genetics

Degree

Ph.D.

Abstract

Among many of the regulators, Rho family GTPases (Rac, Cdc42 and RhoA), PTEN and Myosin 1f have been shown or implicated in the regulation of neutrophil chemotaxis. Yet, the regulatory mechanisms and their functional roles in vivo remain unclear. We propose three specific projects as follows to investigate the functional roles of P-Rex1, PTEN and Myosin 1f in neutrophil chemotaxis. ^ P-Rex1, a guanine nucleotide exchange factor (GEF), was revealed to regulate Rac1 activity. We found that P-Rex1 deficiency caused a decrease of Rac2 activation, but not Rac1 by fMLP. This preference for Rac2 probably resulted from the higher affinity of P-Rex1 for Rac2 than for Rac1. In addition, P-Rex1 deficiency caused a reduction of actin polymerization, a defect in cell motility and a decrease of superoxide formation induced by fMLP. The results suggest that P-Rex1 is a primary Rac2 GEF and regulates Rac2 related neutrophil functions. PTEN has been found repelled from the cell leading edge by active Cdc42 and colocalized at the cell posterior together with active RhoA in activated neutrophils. We found that PTEN posterior membrane localization was impaired, in which chemoattractant-induced RhoA-Rock activation or Pixα,-Cdc42 activation was abrogated in neutrophils. We identified four phosphorylation sites in PTEN, which were shown to be involved in RhoA-Rock mediated PTEN membrane translocation and chemotaxis. In addition, RhoA-Rock regulated PTEN activity and affected PtdIns (3,4,5) level. Cdc42 seemed to regulate PTEN localization through its regulation of RhoA localization. We conclude that RhoA regulates PTEN localization and activity, and Cdc42 regulates PTEN localization probably through RhoA. ^ Myosin 1f, one of actin filament associated molecules, was identified exclusively expressed in mouse neutrophils and its deficiency caused more β2 integrin on the cell surface than that in wild type induced by fibronectin. We found that Myosin 1f deficiency caused a significant reduction of cell chemotaxis on the surface coated with fibronectin, but not with poly-lysine. In addition, Myosin 1f was colocalized with cortical actin, but not with integrin containing granules. We conclude that Myosin 1f may function as a barrier with cortical actin and affect chemotaxis through the regulation of integrin-containing granules release. ^ Keywords. chemotaxis, neutrophils, RhoGTPases, PTEN, Myosin 1f. ^