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First published online 17 January 2006
doi: 10.1242/jcs.02756

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Localised PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ at the phagocytic cup is required for both phagosome closure and Ca²⁺ signalling in HL60 neutrophils

Neutrophil Signalling Group, Wales College of Medicine, Cardiff University, Cardiff, CF14 4XN, UK

^* Author for correspondence (e-mail: hallettmb{at}cf.ac.uk)

Accepted 19 October 2005

Several events accompany integrin-mediated phagocytosis by myeloid cells. These include local pseudopod and phagocytic cup formation followed by Ca²⁺ signalling. However, there is also a role for localised phosphatidylinositol (3,4,5) trisphosphate [PtdIns(3,4,5)P₃] production. Here we report that in neutrophilic HL-60 cells expressing PH-Akt-GFP, binding of iC3b-coated zymosan particles (2 µm in diameter) via ß2 integrin induces an incomplete phagocytic cup to form before either PtdIns(3,4,5)P₃ or phosphatidylinositol (3,4) bisphosphate [PtdIns(3,4)P₂] production or Ca²⁺ signalling. These phosphoinositides then accumulated locally at the site of the phagocytic cup and Ca²⁺ signalling and phagosome closure follows immediately. Although photobleaching showed that PH-Akt-GFP was freely diffusible in the cytosol and able to dissociate from the phagocytic cup, it was restricted to the plasma membrane of the formed but open phagosome and failed to diffuse into the surrounding plasma membrane or neighbouring phagocytic cups even if connected. Inhibition of phosphoinositide (PI) 3-kinase or depletion of membrane cholesterol inhibited both Ca²⁺ signalling and phagosome closure, but had no effect on particle binding or phagocytic cup formation. We therefore conclude that PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ generation was not required for the events that initiate the formation of the phagocytic cup, but that anchoring of PtdIns(3,4,5)P₃ at the phagocytic cup is an essential step for phagosome closure and Ca²⁺ signalling.

Key words: PtdIns(3, 4, 5)P₃, PtdIns(3, 4)P₂, Phagocytosis, Ca²⁺, HL60, Neutrophils

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