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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

View larger version (44K): [in a new window]   Fig. 1. PtdIns(3,4,5)P3 accumulation at the phagocytic cup. (a) Phase-contrast (i and iii) and confocal fluorescent images (ii and iv) of neutrophilic HL60 cells expressing GFP-Akt-PH during phagocytosis. Images i and ii are approximately 30 seconds earlier than images iii and iv, respectively. (b) A series of confocal images of a single phagocytic cup formed around a zymosan particle at the time intervals indicated, showing that GFP-Akt-PH accumulation follows cup formation. The complete data set for this can be seen in Movie 1 of supplementary material. (c) Time course of the intensity of GFP-Akt-PH at the phagocytic cup and at the membrane locations either side (±3 µm). (d) Distribution of GFP-Akt-PH intensity at the membrane in the region of the phagocytic cup, with the boundaries of the phagocytic cup marked. All the images are representative of at least ten similar examples. Bars, 3 µm.

View larger version (51K): [in a new window]   Fig. 2. Three dimensional reconstruction of PtdIns(3,4,5)P3 distribution at the open phagocytic cup. The image series is reconstructed from 20 confocal optical slices through a living cell during phagocytosis, where green shows the high-intensity GFP-Akt-PH binding regions. The reconstruction (left-hand panel) shows an incomplete phagosome that has been sliced through to reveal the inner surface of the open phagocytic cup. The position of the zymosan particle shown in the diagram is also shown in image a. (b) The same data without the zymosan particle. (c and d) The same phagocytic cup reoriented and enlarged to permit visualisation of the inner surfaces of the phagocytic cup. The complete rotation of the 3D reconstruction is shown in Movie 2 in the supplementary material. Bars, 2 µm.

View larger version (57K): [in a new window]   Fig. 3. Distribution of DiI and GFP-Akt-PH. (a) The localisation of exogenously loaded DiIC16(3) to the uropod of a neutrophil. The insert shows the distribution of DiIC16(3) intensity at the membrane in the region of the uropod. (b) The distribution of GFP-Akt-PH (green) and DiIC16(3) (red) in a polarised neutrophilic HL60 cell. (c) The distribution of DiIC16(3) in a neutrophil undergoing phagocytosis. The complete data set for this is shown in Movie 3 of supplementary material. (d) The distribution of GFP-Akt-PH (green) and DiIC16(3) (red) in a neutrophilic HL60 cell during phagocytosis, together with a plot of the intensities of the two probes around the cell periphery and unclosed phagosome. (e) Phase-contrast and confocal fluorescent images of neutrophilic HL60 cells expressing GFP-Akt-PH after treatment with LY294002 (50 µM, 15 minutes, 37°C), showing failure to complete phagocytosis of several iC3b-opsonised zymosan particles and the lack of GFP-Akt-PH translocation to the phagocytic cups (indicated in the phase-contrast image). Bars, 5 µm.

View larger version (92K): [in a new window]   Fig. 4. Mobility of PtdIns(3,4,5)P3 in neutrophilic cells. The effects of localised photobleaching on GFP-Akt-PH. In each data set, the first image shows the distribution of GFP-Akt-PH before laser photobleaching, the middle image is the zone of localised bleaching marked by the rectangle and the last image shows the effect 30 seconds after bleaching in a resting neutrophilic HL60 cell (a), a neutrophilic HL60 cell undergoing phagocytosis (b) and a neutrophilic HL60 cell with a completed phagosome (c). These data are typical of at least three similar experiments in each case. Bars, 2 µm.

View larger version (46K): [in a new window]   Fig. 5. Cholesterol depletion disrupts PtdIns(3,4,5)P3 accumulation. (a) Distribution of filipin staining in fixed neutrophils before and after treatment with methyl-ß-cyclodextrin. (b) Quantification of the reduction in `peripheral' or membrane staining in neutrophils before and after treatment with methyl-ß-cyclodextrin. The data are shown as the percentage of filipin staining at the cell periphery. (c) Images of a neutrophilic HL60 cell treated with methyl-ß-cyclodextrin demonstrating the failure of GFP-Akt-PH to accumulate at phagocytic cups. The cell was stained with DiIC16(3) (red) and GFP-Akt-PH (green) with the position of two zymosan particles indicated. This cell was typical of at least ten others. Bars, 4 µm.

View larger version (61K): [in a new window]   Fig. 6. PtdIns(3,4,5)P3 generation at individual phagocytic cups. (a) A micropipette was used to present two iC3b-opsonised zymosan particles, (labelled Z1 and Z2) to a neutrophilic HL60 cell shown in the phase-contrast image and the simultaneously acquired confocal image of the distribution of GFP-Akt-PH (green). The fluorescent images from the zone indicated by the rectangle are shown below at 10 second intervals, with the location of particles Z1 and Z2 indicated on the first image. The complete data set is shown as a movie (Movie 4 in supplementary material) and the time course of GFP-Akt-PH accumulation in the membranes at these two locations is shown in b. Example of the distribution of GFP-Akt-PH in two phagosomes sequentially formed and touching (c) and simultaneously forming at opposite poles of the cell (d). In c and d the images show GFP-Akt-PH distribution at 20 second intervals. Bars, 2 µm.

View larger version (52K): [in a new window]   Fig. 7. Inhibition of Phagocytic cup closure by PI 3-kinase inhibition and cholesterol depletion. The failure to complete phagocytosis and the distribution GFP-Akt-PH (green) is shown after treatment with LY294004 (a) and methyl-ß-cyclodextrin (b). The incomplete phagocytic cups are indicated in a by arrows and the position of a single zymosan particle indicated in b by the white circle. A plot of the intensity of peripheral GFP-Akt-PH is shown below b with the position of the incomplete phagocytic cup indicated by the double-headed arrow. (c) Histograms of completed phagocytosis (darker shading) and the formation of phagocytic cups (lighter shading) 20 minutes after micromanipulated delivery of iC3b-opsonised zymosan particles to (i) neutrophils and (ii) neutrophilic HL60 cells untreated (control) or treated with LY294004 (LY) and methyl-ß-cyclodextrin (MßCD) as a percentage of deliveries. There was no significant difference between treatments in the total of incomplete and complete phagocytosis (both shadings in a single histogram column) showing that binding and phagocytic cup formation were not affected by the treatments, whereas the percentage of failed phagocytosis which arrested at the stage of phagocytic cup formation (lighter shading) was increased by the treatments. Bars, 3 µm.

View larger version (46K): [in a new window]   Fig. 8. Phagocytic PtdIns(3,4,5)P3 accumulation and Ca2+ signalling. (a) Traces i and ii are typical examples of cytosolic free-Ca2+ oscillations after contact between iC3b-opsonised zymosan particles and neutrophilic HL60 cells. (b) The upper trace (labelled PIP3) shows the intensity of GFP-Akt-PH at the phagocytic cup and the lower trace (labelled Ca2+) cytosolic free-Ca2+ concentration measured simultaneously within an individual neutrophilic HL60 cell, using fluo4 as Ca2+ indicator. The three images (left to right) show: (1) the accumulation of PtdIns(3,4,5)P3 at the phagocytic cup (GFP-Akt-PH at the membrane) before an increase in cytosolic free Ca2+ (fluo4 intensity in the cytosol); (2) Ca2+ (fluo4 intensity in the cytosol) elevation before phagosome closure; and (3) the phagosome is closed and the first Ca2+ spike subsides. The complete data set can be seen in Movie 5 of the supplementary material, in which the subsequent cytosolic free-Ca2+ spikes are also shown. (c) Sequence of phase-contrast images of a representative example of the micromanipulated presentation of iC3b-opsonised zymosan particles to neutrophils after treatment with methyl-ß-cyclodextrin. The three images show contact, binding, and formation of a weak phagocytic cup (left to right). The lower trace shows the lack of change in cytosolic free Ca2+ in the cell during the experiment. (d) A sequence of the accumulation of GFP-Akt-PH at the point of firm contact between an iC3b-opsonised particle and a neutrophilic HL60 cell pretreated with cytochalasin B (5 µg/ml, 5 minutes) to prevent phagocytic cup formation. The complete data set is also shown in Movie 6 of supplementary material. Neither mechanical distortion of the membrane using a naked micropipette tip nor firm contact with un-opsonised zymosan particle elicited GFP-Akt-PH accumulation. Bars, 3 µm.