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Figure 8


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.





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