Abstract
Class I phosphoinositide 3-kinases (PI3Ks) are important regulators of neutrophil migration in response to a range of chemoattractants. Their primary lipid products PtdIns(3,4,5)P3 and PtdIns(3,4)P2 preferentially accumulate near to the leading edge of migrating cells and are thought to act as an important cue organizing molecular and morphological polarization. We have investigated the distribution and accumulation of these lipids independently in mouse neutrophils using eGFP-PH reportersand electron microscopy (EM).We found that authentic mouse neutrophils rapidly polarized their Class I PI3K signalling, as read-out by eGFP-PH reporters, both at the up-gradient leading edge in response to local stimulation with fMLP as well as spontaneously and randomly in response to uniform stimulation. EM studies revealed these events occurred at the plasma membrane, were dominated by accumulation of PtdIns(3,4,5)P3, but not PtdIns(3,4)P2, and were dependent on PI3Kγ and its upstream activation by both Ras and Gβγs.
Highlights
Neutrophils are key players in the innate anti-bacterial and fungal defence mechanisms of mammals
Expression of membrane-targeted-FP constructs has indicated that membrane accumulation near to the leading edge can contribute to the apparent polarization of Pleckstrin homology (PH) domain reporters (Dewitt et al, 2009; Onsum et al, 2006)
We tested to what extent phenomenon impacted our observations by co-expressing of mCherry-CAAX with enhanced Green Fluorescent Protein (eGFP)-PH-Protein Kinase B (PKB) in mouse neutrophils and analysing their distribution during chemotaxis towards formylated peptides (Fig. 2A)
Summary
Neutrophils are key players in the innate anti-bacterial and fungal defence mechanisms of mammals They are capable of exiting the circulation in the proximity of local infections or tissue damage and chemotaxing towards the epicentre of the inflammatory response. This work has identified a large number of intracellular signals and/or proteins upon which neutrophil chemotaxis can depend Amongst these phosphoinositide 3-kinases (PI3Ks) (Hirsch et al, 2000; Li et al, 2000; Sasaki et al, 2000) and Rac-family GTPases (Roberts et al, 1999; Servant et al, 2000; Weiner et al, 2002) have been implicated repeatedly but other key regulators include Ca2þ (Evans and Falke, 2007) and p38 MAPKs (Heit et al, 2002). PtdIns5P has been implicated in cell migration (Viaud et al, 2014 #3612) there is no evidence establishing that it has a role in neutrophil chemokinesis (Bulley et al, 2015)
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