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* Division of Medical Microbiology, Institute for Molecular and Clinical Medicine and
Division of Cell Biology, Institute for Biomedicine and Surgery, Faculty of Health Sciences, Linköping University, Linköping, Sweden
Phagocytosis is a complex process involving the activation of various signaling pathways, such as the Rho GTPases, and the subsequent reorganization of the actin cytoskeleton. In neutrophils, Rac and Cdc42 are activated during phagocytosis but less is known about the involvement of these GTPases during the different stages of the phagocytic process. The aim of this study was to elucidate the role of Cdc42 in phagocytosis and the subsequent phagosomal maturation. Using a TAT-based protein transduction technique, we introduced dominant negative and constitutively active forms of Cdc42 into neutrophil-like HL60 (human leukemia) cells that were allowed to phagocytose IgG-opsonized yeast particles. Staining of cellular F-actin in cells transduced with constitutively active Cdc42 revealed that the activation of Cdc42 induced sustained accumulation of periphagosomal actin. Moreover, the fusion of azurophilic granules with the phagosomal membrane was prevented by the accumulated F-actin. In contrast, introducing dominant negative Cdc42 impaired the translocation per se of azurophilic granules to the periphagosomal area. These results show that efficient phagosomal maturation and the subsequent eradication of ingested microbes in human neutrophils is dependent on a strictly regulated Cdc42. To induce granule translocation, Cdc42 must be in its active state but has to be inactivated to allow depolymerization of the F-actin cage around the phagosome, a process essential for phagolysosome formation.
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1 This work was supported by grants from the King Gustaf V Memorial Foundation, the Swedish Research Council (13103 and 5968), the Swedish Medical Society, and the Medical County Council of Östergötland. M.L. was supported by a postdoctoral fellowship from the Swedish Society for Medical Research and an assisting professor fellowship from the Swedish Research Council (529-2003-5994), V.P.D. by a Ph.D. student fellowship from the Network for Inflammation Research funded by the Swedish Foundation for Strategic Research, and E.S. by an assisting professor fellowship from the Swedish Medical Research Council (12725).
2 M.L. and V.P.B. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Eva Särndahl, Division of Medical Microbiology, Faculty of Health Sciences, Linköping University, SE-58185 Linköping, Sweden. E-mail address: eva.sarndahl{at}imk.liu.se
4 Abbreviations used in this paper: PMB, polymyxin B; HA, hemagglutinin; KRG, Krebs-Ringer phosphate buffer; N17Cdc42, dominant negative form of Cdc42; PFA, paraformaldehyde; RSD, relative SD; V12Cdc42, constitutively active form of Cdc42.
5 The online version of this article contains supplemental material.
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