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

Neutrophils and neutrophilic HL60s can bind and internalise microscopic particles (with diameters of 0.5-3.0 μm) of a number of different surface materials. However, coating the surface of the particles with opsonins, such as the complement component iC3b, increases the binding and the speed of subsequent phagocytosis. The iC3b-accelerated phagocytosis of infecting microorganisms by neutrophils is probably the main route for the first line of defence by the innate immune system in vivo and is also involved in antibody-mediated phagocytosis (Jongstra-Bilen et al., 2003). Surface-bound iC3b binds the complement receptor CR3 (CD18/CD11b) which is a β2-integrin, resulting in an intricate interplay with Ca²⁺ signalling. Not only does immobilisation of β2-integrin on the neutrophil surface trigger cytosolic free-Ca²⁺ signalling (Ng-Sikorski et al., 1991; Jaconi et al., 1991; Petersen et al., 1993; Pettit and Hallett, 1996; Dewitt and Hallett, 2002), but changes in cytosolic free-Ca²⁺ concentration also signal outwards to integrins, increasing the effectiveness of the receptor (Dewitt and Hallett, 2002; Pettit and Hallett, 1998). We have recently shown that locally engaged β2-integrin triggers a complex Ca²⁺ signal which initially causes the release of β2 integrin molecules that were cytoskeletally tethered and so contributes to the acceleration process (Dewitt and Hallett, 2002). Subsequent components of the Ca²⁺ signal activate the phagosomally assembled oxidase system (Dewitt et al., 2003).

Although the engagement of β2 integrins also activates PI 3-kinase activity (Axelsson et al., 2000) it is less clear how this relates to these events. It is known that PtdIns3P is formed on the membrane of the closed phagosome (Stephens et al., 2002) and acts a signal for oxidase assembly through p47phox (Ellson et al., 2001) and p67phox (Kanai et al., 2001) binding via a PX domain (Ellson et al., 2001; Kanai et al., 2001). Grinstein and colleagues have also shown that PtdIns(4,5)P₂ is generated at the site of phagocytosis in macrophages early in the process of phagosome formation, as a result of activation of PtdIns(4,5)P₂ kinase (Botelho et al., 2000; Coppolino et al., 2002) and that PtdIns(1,4,5)P₃ is generated at the same site during Fcγ R (antibody-mediated) phagocytosis by macrophages (Vieira et al., 2001; Marshall et al., 2001). PtdIns(4,5)P₂ is a source of Ins(1,4,5)P₃ and diacylglycerol and also acts with the Ca²⁺-activated protein gelsolin to cause actin nucleation and de-polymerisation. The role of PtdIns(1,4,5)P₃ or PtdIns(4,5)P₂ is less clear but they do have an important role in determining the polarity of myeloid cells, with PtdIns(1,4,5)P₃ localising to the leading edge of neutrophilic HL60 cells during chemotaxis (Servant et al., 2000; Weiner et al., 2002; Xu et al., 2003; Srinivasan et al., 2003). This location is maintained by its local generation and destruction giving a finely and spatially tuned distribution (Xu et al., 2003; Srinivasan et al., 2003). There is evidence that PtdIns(3,4,5)P₃ generation in the membrane of macrophages during phagocytosis permits pseudopod extension, especially around larger particles (Cox et al., 2002) and that this effect may be mediated by acting through the PH-domain-containing cytoskeletal protein myosin X (Cox et al., 2002; Berg et al., 2000).

To further our understanding of the role of PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ during phagocytosis in myeloid neutrophilic cells, it is important to establish when PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ generation and Ca²⁺ signalling occurs during the sequence of events which comprise phagocytosis. The aim of the work reported here, therefore, was to visualise the dynamic PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ generation during iC3b-mediated phagocytosis by neutrophilic HL60 cells and to correlate this with particle binding, phagocytic cup formation, phagosome closure and Ca²⁺ signalling. We report that binding and phagocytic initiation occurs without a requirement for PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ accumulation in the membrane or Ca²⁺ signalling. However, PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ accumulation and `anchoring' at the phagocytic membrane is required for both Ca²⁺ signalling and completion of phagocytosis by phagosome closure.

The expression of PH-Akt-GFP had no effect on the ability of HL60 neutrophils to undergo phagocytosis when challenged with iC3b-opsonised zymosan particles. An extremely localised accumulation of PH-Akt-GFP occurred at sites of iC3b-mediated phagocytosis consistent with the production of PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ (Fig. 1). It was clear that iC3b-mediated zymosan binding and phagocytic cup formation occurred prior to PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ accumulation (Fig. 1b). PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ accumulated at the phagocytic cup membrane after its formation. No localised accumulation of PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ was observed at membranes sites before formation of the phagocytic cup. It was important to observe the sequence in real time as once PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ accumulation had occurred at the phagocytic cup, the cup subsequently rapidly proceeded to closure (see e.g. Fig. 1a). This sequence (i.e. binding of the iC3b opsonised particles and formation of the phagocytic cup before PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ generation) is shown in Fig. 1b, Fig. 6a and in Movie 1 in supplementary material. In all cases (see Fig. 1b, Fig. 6a), the membrane delimiting the phagocytic cup initially formed around the zymosan particle with the same PH-Akt-GFP intensity as the bulk cytosol, indicating that there was no accumulation of the probe at the phagocytic cup membrane. Subsequently, and without further change in the curvature or extent of contact between the cell and the particle, PH-Akt-GFP accumulation in the cup membrane then occurred.

The accumulation of PH-Akt-GFP was localised to the phagocytic cup, with no increased fluorescence appearing in the surrounding membrane near the open phagosome (Fig. 1a,b,c). Three-dimensional visualisation showed that PH-Akt-GFP accumulated over the complete surface of the phagocytic cup and in no optical plane could PH-Akt-GFP accumulation be observed outside the unclosed phagosome (Fig. 2, Movie 2 in supplementary material). In order to establish whether this extremely localised increase in PH-Akt-GFP was the result of a localised increased membrane ruffling or unfolding or a partitioning effect not related to PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ production, the membranes of PH-Akt-GFP-expressing cells were also visualised with DiIC₁₆(3). This label was chosen as: (1) it is also capable of reporting extreme localisation, for example in the well-defined uropod of polarised neutrophils (Fig. 3a); and (2) its location in the cell can be independent of PH-Akt-GFP distribution; PH-Akt-GFP being located towards the front in the same cell in which DiIC₁₆(3) was located towards the rear (in the less well-defined uropod of neutrophilic HL60 cells) (Fig. 3b). In neutrophils, DiIC₁₆(3) was neither excluded nor accumulated at the phagocytic cup (Fig. 3c and Movie 3 in supplementary material). Similarly in neutrophilic HL60 cells, PH-Akt-GFP accumulated at the phagocytic cup without any accompanying accumulation or exclusion of DiIC₁₆(3) (Fig. 3d). As the accumulation of GFP-PH-Akt fluorescence was also totally inhibited by pretreatment with the PI 3-kinase inhibitor LY294002 (50 μM, 15 minutes, 37°C; Fig. 3e), GFP-PH-Akt localisation was attributed solely to the local generation of PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ and not to a change in membrane ruffling or other 3D effect at the plane of imaging.

As there is evidence that PtdIns(3,4,5)P₃ production is involved in pseudopodia extension, it was noteworthy that during accumulation of PH-Akt-GFP at the phagocytic cup there was no evidence of an elevation in PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ at the pseudopod extensions of the cup or even in the membrane within 3 μm of the cup (Fig. 1c, Fig. 2). Furthermore, there was a sharp boundary between the PH-Akt-GFP in the phagocytic cup and membrane zones either side (Fig. 1a,d), with no detectable lateral diffusional leakage of PH-Akt-GFP across this boundary. The possibility existed that PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ was physically unable to move out of the phagocytic cup because it was `locked onto' the phagosomal cytoskeleton. However, this seemed unlikely as localised photobleaching showed that cytosolic PH-Akt-GFP was freely diffusible (Fig. 4a; D=∼20 μm²/second for GFP-PH-Akt compared with 25 μm²/second for GFP alone) (see Dayel et al., 1999), and PH-Akt-GFP at the phagocytic cup (Fig. 4b) or formed phagosome (Fig. 4c) were also free to dissociate. Photobleaching at any location within the neutrophilic HL-60 cell resulted in a decrease in PH-Akt-GFP intensity at the phagocytic cup or complete phagosome (Fig. 4b), indicating that not only was PH-Akt-GFP mobile in the cell, but that its dissociation rate from its PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ binding location was reasonably fast (t_1/2 was estimated to be less than 30 seconds).

The demonstration by photobleaching that GFP-Akt-PH was free to diffuse and dissociate made it unlikely that the lack of lateral movement of GFP-Akt-PH at the phagocytic cup membrane was either due an interaction of GFP-Akt-PH with non-PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ immobile elements in the cell or `trapping' in the peri-phagosomal actin network. As there is evidence that PtdIns(3,4,5)P₃ and PtdIns(3,4,5)P₃-GFP-Akt-PH complex may be restricted into polyphosphoinositol-containing lipid rafts which are disrupted by membrane cholesterol depletion (Gomez-Mouton et al., 2004; Seveau et al., 2001), the effect of cholesterol depletion on PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ localisation was examined. Pre-treatment of neutrophils with methyl-β-cyclodextrin significantly reduced the membrane binding of the cholesterol marker filipin (Fig. 5a,b). This treatment also totally inhibited PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ localisation during phagocytosis by HL60 neutrophilic cells (Fig. 5c), suggesting that cholesterol may be an element in the restriction of PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂.

Since the lateral diffusion of GFP-Akt-PH was restricted to the phagocytic cup, this gave rise to the possibility of visualising individual sites of PI 3-kinase activity. The generation of PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ within the phagocytic cup usually spread from only one (or sometimes two) sites within the cup (see e.g. Fig. 1b and Movie 1 in supplementary material), suggesting that there was only a small number of foci of PI 3-kinase activity within a phagosome. PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ production usually originated from only one locus per phagocytic cup and there was a marked asynchrony between PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ production in phagocytic cups when forming around more than one particle (Fig. 6a). Even when the two phagocytic cups were adjacent, i.e. within 2 μm of each other (Fig. 6a,b), or a second phagocytic cup formed touching the first (Fig. 6c), there was no diffusion of PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ from one open cup to another (Fig. 6c). The asynchrony of PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ generation between phagocytic cups was unlikely to be due to local depletion of either the substrate PtdIns(3,4)P₂ or the enzyme PI 3-kinase by one of the two phagocytic cups because the same asynchrony was observed in phagocytic cups at opposite poles of the cell, i.e. at least 15 μm apart (Fig. 6d). Instead, it may simply reflect the stochastic nature of very small numbers of foci of PI 3-kinase activity.

Inhibition of localised PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ accumulation by either inhibition of PI 3-kinase activity (LY294002) or by cholesterol depletion (methyl-β-cyclodextrin) did not have a significant effect on the binding of iC3b-opsonised particles presented to the cells by micropipette delivery or on the subsequent formation of phagocytic cups. However, these inhibitors had a profound inhibitory effect on phagocytosis. The progress to phagosome closure was severely inhibited (Fig. 7a,b,c) with phagocytic cups rarely proceeding to closure. The link between prevention of PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ localisation and the formation of incomplete phagocytic cups was consistent with a role for PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ in the extension of the pseudopodia around attached particles (Cox et al., 1999).

It was important to establish whether the obligatory role of PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ accumulation for phagosome closure was upstream or downstream of cytosolic free-Ca²⁺ signalling, as a similar arrest of phagocytosis can be achieved by inhibition of Ca²⁺ signalling or the Ca²⁺-activated enzyme, calpain (Dewitt and Hallett, 2002; Dewitt et al., 2003). The possibility that PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ production was upstream of Ca²⁺ signalling existed because PtdIns(3,4,5)P₃ may activate PLC-γ and Ins(1,4,5)P₃ production (Rameh et al., 1998) or be independent of phospholipase activation (Pasquet et al., 2000). Furthermore in neutrophils, the addition of exogenous PtdIns(3,4,5)P₃ to the inner leaflet of the plasma membrane bilayer by itself elicits oscillatory Ca²⁺ signals (Tian et al., 2003). In neutrophilic HL60 cells, iC3b stimulation similarly provoked large oscillatory Ca²⁺ signals which were irregular in size and frequency and varied from cell to cell (Fig. 8a,b and movie 5). These characteristics were similar to the iC3b-β2-integrin-induced Ca²⁺ signals observed in human neutrophils (Dewitt and Hallett, 2002; Dewitt et al., 2003). No evidence of regular Ca²⁺ spikes or ultra-fast cortical Ca²⁺ `waves' recently reported (Worth et al., 2003; Kindzelskii and Petty, 2003) were found. The Ca²⁺ signalling event in neutrophilic HL60 cells was triggered after the onset of phagocytic cup formation but before its closure, and continued to oscillate after phagosome closure (Fig. 8a). The event window during which Ca²⁺ signalling was triggered was therefore similar to that observed in human neutrophils (Dewitt and Hallett, 2002; Dewitt et al., 2003) and was within the time frame at which PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ was accumulating in phagocytic cups. The temporal relationship between PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ formation and the Ca²⁺ signalling was established within the same cells by monitoring PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ generation (PH-Akt-GFP accumulation) and cytosolic free Ca²⁺ simultaneously. In all cells examined, PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ generation at the phagocytic cup preceded the onset of the Ca²⁺ signal (Fig. 8b and Movie 5 in supplementary material) by between 20-100 seconds, but both always occurred before phagosome closure. The cytosolic free-Ca²⁺ signal was therefore an event later than both phagocytic cup formation and PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ formation but before phagosome closure. Prevention of PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ accumulation by the PI 3-kinase inhibitor LY294002 or cholesterol depletion also inhibited the accompanying Ca²⁺ signalling in HL60 neutrophilic cells or neutrophils (Fig. 8c). As triggering of the Ca²⁺ signal depends on a crucial number of β2 integrin-iC3b molecular pairings which result during extension of the cups of the phagocytic cup (Dewitt and Hallett, 2002), any experimental procedure which prevents or reduces the extent of phagocytic cup formation may prevent delivery of the threshold stimulus (and hence its Ca²⁺ signal read-out) to be lost. However, this was unlikely to provide the explanation for inhibition of the Ca²⁺ signal because blocking the phagocytic cup formation by prevention of actin polymerisation (cytochalasin B pre-treatment) failed to inhibit Ca²⁺ signalling in neutrophils (Dewitt and Hallett, 2002; Dewitt et al., 2003) and PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ accumulation at the iC3b-zymosan contact site (Fig. 8d). It was therefore concluded that PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ generation and its localisation at the phagocytic cup is necessary for both phagosome completion and Ca²⁺ signalling.

The data presented here demonstrate that in neutrophilic cells, binding of PH-Akt-GFP occurred after phagocytic cup formation where it is restricted before phagosome closure. Since PH-Akt-GFP binds PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂, signalling via Akt is thus a late signalling event relative to particle binding via β2 integrin and formation of the phagocytic cup, but is earlier than Ca²⁺ signalling and closure of the cup. This data shows that PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ restriction at the phagocytic cup were essential for both progression of the cup and phagosome closure and cytosolic free Ca²⁺ signalling.

These data raise two main series of questions. The first questions concern the mechanism by which PH-Akt-GFP-PtdIns(3,4,5)P₃ is restricted at the phagocytic cup site. We have excluded the possibility that PH-Akt-GFP anchorage was in some way artefactually generated and not a reflection of PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ distribution. Photobleaching of GFP showed that PH-Akt-GFP was free to diffuse in the cytosol and readily able to dissociate from the phagocytic cup. Thus the restricted distribution of PH-Akt-GFP to the phagocytic cup was not a property of an immobility of PH-Akt-GFP. As PH-Akt-GFP accumulation was also prevented by inhibition of PI-3-kinase, we conclude that PH-Akt-GFP accurately marked PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ and that the mobility of PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ was extremely restricted. Exogenously incorporated PtdIns(3,4,5)P₃ also becomes immobilised at the plasma membrane of neutrophils and is ultimately cleared to the uropod (Weiner et al., 2002; Tian et al., 2003). The data here suggests that cholesterol plays a role in the restriction of PtdIns(3,4,5)P₃. Cholesterol-rich lipid-rafts have been shown that form often at the uropod or other locations both in neutrophils (Seveau et al., 2001) and in neutrophilic HL60 (Gomez-Mouton et al., 2004). The lipid raft itself may be immobilised by PIP₂ and PtdIns(3,4,5)P₃ molecules acting as cytoskeletal anchorage points. There are at least two candidate molecules for PtdIns(3,4,5)P₃-cytoskelton linkage: (1) myosin-X, which has a PtdIns(3,4,5)P₃-binding PH domain and may act as a link from PtdIns(3,4,5)P₃-containing regions of the plasma membrane to the cytoskeleton (Cox et al., 2002; Berg et al., 2000); and (2) WAVE-2, the Arp2/3 complex activator which binds PtdIns(3,4,5)P₃ independently of PH-domains (Oikawa et al., 2004). In a monocytic cell line PtdIns(3,4)P₂ accumulates transiently at the site of phagocytosis and is lost before the phagosome seals (Coppolino et al., 2002). It is unlikely that the loss of PtdIns(3,4)P₂ from the phagocytic cup would be result from its consumption to generate PtdIns(3,4,5)P₃ (via PI 3-kinase). It is also unlikely from our data that PtdIns(3,4)P₂ was broken down to release Ins(3,4,5)P₃ early in the formation of the phagocytic cup, as the cytosolic free Ca²⁺ signal, which is a read-out for Ins(3,4,5)P₃ production, was detected only after significant phagocytic cup formation and persisted after phagosome closure (see Fig. 8).

The second series of questions relate to the role of PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ in phagosome closure. The data here has shown that inhibition of PtdIns(3,4,5)P₃- or PtdIns(3,4)P₂-localised accumulation at the phagocytic cup results in inhibition of both phagosome closure and its associated Ca²⁺ signal. As these latter two events are interconnected, care must be taken in drawing a conclusion. The Ca²⁺ signal is triggered only when a threshold number of integrin-iC3b molecules are engaged. A suitably large contact area between the particle and the cell within the forming phagocytic cup must form to produce this threshold number of engagements. This means that strategies that reduce the extent of phagocytic cup formation could also inhibit the Ca²⁺ signal. However, this is unlikely to account for the inhibition of the Ca²⁺ signal by inhibition of PI 3-kinase or PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ accumulation because (1) in the absence of PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ accumulation, phagocytic cups formed which were normally large enough to provoke a Ca²⁺ signal and (2) when phagocytic cup formation was prevented by cytochalasin B, PtdIns(3,4,5)P₃ and Ca²⁺ signalling was provoked in the absence of a phagocytic cup provided that the contact area between the particle and the cell was increased by micromanipulation (see Fig. 8). It may therefore be concluded that PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ localisation was a prerequisite for generation of the Ca²⁺ signal rather than vice versa. The mechanism for the link between PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ generation and Ca²⁺ signalling is unclear, but may involve a similar mechanism to that described in platelets (Pasquet et al., 2000) and involve activation of phospholipase C γ. Interestingly, it has been shown that to reconstruct the signalling to the neutrophil oxidase in COS cells, it is necessary to also express PKC δ, phospholipase C-β2 and the PtdIns(3,4,5)P₃-generating enzyme, PI 3-kinase γ (He et al., 2004). A causal link between PtdIns(3,4,5)P₃ and Ca²⁺ signalling would also provide an explanation for its role in phagosome formation. The phagocytic Ca²⁺ signal is important for accelerating phagocytosis by activating calpain (Dewitt and Hallett, 2002), and inhibition of this system arrests phagocytosis at the stage of phagocytic cup.

The data presented here is thus consistent with a causal role of PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ in phagosome closure but not binding or phagocytic cup formation. Firstly, PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ generation and restriction occurs after phagocytic cup formation but precedes phagosome closure (Fig. 1a,b, Fig. 2a, Fig. 4b). Second, inhibition of PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ formation and accumulation did not prevent phagocytic cup formation, but did inhibit phagosome closure (Fig. 3e, Fig. 5c, Fig. 7a,b,c, Fig. 8d). This latter finding has also been reported in macrophages, where treatment with LY294002 also arrested phagosome formation before closure, but after phagocytic cup formation (Cox et al., 1999; Araki et al., 1996). In these cells, internalisation of particles of different sizes has elegantly demonstrated that pseudopod extension around the particle, rather than binding or signalling, is the key PI 3-kinase-dependent step (Cox et al., 1999). Although PtdIns(3,4,5)P₃ generation and location in macrophages were not visualised in these earlier studies, the data here confirm the involvement of PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ in the phagocytic cup with the process of phagosome closure. The locations of PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ which we observe, however, do not support the idea that that they are involved in extending pseudopodia by addition of PtdIns(3,4,5)P₃- or PtdIns(3,4)P₂-containing membrane to the pseudopod tips. As well as its role in Ca²⁺ signalling, it is however possible that immobilisation of the PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ to the cytoskeleton in the phagocytic cup provides a leverage and anchorage point from which actin is polymerised and pseudopodia are extended around the particle (Cox et al., 2002; Zhang et al., 2004).

PH-Akt-GFP expressing HL-60 cells, derived as previously described (Servant et al., 2000; Weiner et al., 2002; Xu et al., 2003; Srinivasan et al., 2003) were kindly supplied by H. R. Bourne, UCSF, CA. Undifferentiated PH-Akt-GFP expressing HL60 cells were grown in RPMI 1640 supplemented with 25 mM HEPES, 10% fetal bovine serum, 100 μg/ml streptomycin, 100 U/ml penicillin and 2 mM L-glutamine. They were differentiated by treatment of cells (10⁵ cells/ml) with DMSO (1.3% v/v) for 6 or 7 days with additional medium added at day 4. Under these conditions, differentiation was optimal as determined by expression of CD11b (the CR3 receptor for iC3b), adherence to glass coverslips and phagocytosis of C3bi-opsonised zymosan. PH-Akt-GFP binds to PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ and hence its distribution within the cells marks the locations at which PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ are elevated relative to others.

Neutrophils loaded with 1,1′-dihexadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate [DiIC₁₆(3)] (Molecular Probes) or PH-Akt-GFP in HL-60 neutrophilic cells were imaged confocally using rapid resonant scanning (Leica RS) during phagocytosis. Phase-contrast images were acquired simultaneously. The 3D data were acquired by rapid z-sectioning in living cells during phagocytosis. Up to 20 z sections through the cell undergoing phagocytosis were taken without distortion provided that no cell movement occurred between the first and last sections. The 3D reconstructed images of the GFP-Akt distribution were rendered using Imaris software (Biplane). The distribution of GFP or DiI at the cell periphery was quantified using ImageTool (University of Texas Health Science Center) and expressed relative to the mean fluorescence intensity of the entire periphery.

Neutrophilic HL60 cells were loaded with either Fluo4 or Fura-red from their acetoxy-methyl esters (Molecular Probes) by incubation at 37°C with the probe dissolved in dry DMSO plus Pluronic (20% w/v) to give cytosolic concentrations of the acid form of the probe of approximately 50 μM. This had no effect on the polarization or phagocytic capacity of the cells and was expected to increase the Ca²⁺-buffering capacity of the cells by less than 10%. When used in cells expressing PH-Akt-GFP, the distribution of fluorescent signal was used as an indicator of PtdIns(3,4,5)P₃ or PtdIns(3,4)P₂ accumulation in the membrane, whereas cytosolic fluorescence intensity was used as a separate readout for cytosolic free-Ca²⁺ concentration.

The mobility of GFP-PH-Akt within neutrophilic HL60 cells was assessed by photobleaching areas of the cell at a confocal plane that approximately bisected the phagosome to provide an image of the phagosomal equator at low laser power. The voltage on the photomultiplier tube was set to maximum and line averaging was used. The part of the cell to be photo-bleached was subjected to a high power laser-scanning pulse (30 seconds). The post photobleaching confocal images were then acquired at the original laser power setting.

Zymosan particles (10 mg/ml) were opsonised either by incubation with human serum (50% diluted, 30 minutes, 37°C). The particles were then washed by centrifugation and resuspension to remove unfixed C3bi and either used immediately or stored at –20°C. Neutrophilic HL60 cells adherent to glass coverslips were mounted for confocal imaging. Zymosan particles were allowed to sediment among the cells. Phagocytosis of the particles close to the neutrophilic HL60 cells occurred spontaneously. An alternative approach that negated the requirement for spontaneous pseudopod formation by neutrophilic HL60 cells, was to draw a single particle into the mouth of the micropipette (1 μm tip diameter) by applying slight negative pressure, and lightly placing the particle in contact with a neutrophilic HL60 cell as described previously (Dewitt and Hallett, 2002; Dewitt et al., 2003). After adherence between cell and particle was established, the zymosan was released by removing the negative pressure and phagocytosis allowed to proceed (Dewitt and Hallett, 2002; Dewitt et al., 2003). The method of particle presentation made no difference to any of the events described here.

Methyl-β-cyclodextrin, (MβCD, Sigma) was used to deplete neutrophils and HL-60 neutrophilic cells of membrane cholesterol. Fresh stock solutions of MβCD were prepared in RPMI medium or Krebs for HL-60 or primary neutrophils respectively, for each experiment. Cells were incubated with MβCD (10 mM) for 60-90 minutes at 37°C. Cholesterol depletion was monitored by filipin binding (0.05 mg/ml, with 10% BSA, 1 hour at room temperature) to paraformaldehyde (4%) fixed cells. Cells were washed three times with PBS and incubated with glycine (1.5 mg/ml, 20 minutes at room temperature) to quench paraformaldehyde fluorescence. Fluorescence was visualised and measured non-confocally by excitation at 360 nm (emission 410-500 nm). The plasma-membrane-associated fluorescence was quantified by determining the peripheral fluorescence as a percentage of the total cellular fluorescence.

We are grateful to Henry R. Bourne (UCSF) for kindly supplying the PH-Akt-GFP expressing HL60 cell line used in this study and for support from the Wellcome Trust (UK) to S.D. and W.T.