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First published online 30 November 2004
doi: 10.1242/jcs.01579

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In vivo analysis of 3-phosphoinositide dynamics during Dictyostelium phagocytosis and chemotaxis

¹ Division of Cell and Developmental Biology, MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH, UK
² BioFocus Limited, Milton Road, Cambridge, CB4 0FG, UK

View larger version (23K): [in a new window]   Fig. 1. The PI3-kinase inhibitor LY294002 affects phagocytosis. (A) Wild-type AX3 cells were incubated with TRITC-labelled yeast and at the indicated time points the fluorescence of internalised particles was measured. Average and standard deviation of three independent experiments are shown. (B) Uptake of fluorescent 1 µm beads by AX3 cells. Average and standard deviation of three independent experiments are shown. (C) Effect of LY294002 treatment on phagocytosis of bacteria by AX3 cells. The internalisation of bacteria was measured as a reduction of the optical density at 600 nm, here plotted as a percentage of the OD600 at t=0 hours. The curve `bacteria' refers to a control with bacteria but without Dictyostelium cells. The average and standard deviation of three independent experiments are shown.

View larger version (61K): [in a new window]   Fig. 2. Lipid binding specificity of PH domain GFP fusion proteins. Protein lipid overlay assays were used to assess the lipid binding specificities of the various probes. Phosphoinositides (200 pmol) were spotted on nitrocellulose membranes, which were incubated with extracts from cells expressing the PH-GFP fusion proteins. Binding is directly detected using the GFP fluorescence. (A,B) Qualitative assays to determine binding specificity of CRAC, GRP1 and TAPP1. CRAC binds to both PtdIns(3,4,5)P3 and PtdIns(3,4)P2, while GRP1 only recognises PtdIns(3,4,5)P3 and TAPP1 is specific for PtdIns(3,4)P2. (C) Lipid dilution series to compare relative affinities of DAPP1-PH and GRP1-PH for PtdIns(3,4,5)P3 and PtdIns(3,4)P2. GRP1-PH shows strong binding even to low levels of PtdIns(3,4,5)P3, while appreciable DAPP1-PH binding only occurs at high lipid concentrations. Note that DAPP1 binds to both PtdIns(3,4,5)P3 and PtdIns(3,4)P2.

View larger version (101K): [in a new window]   Fig. 3. Localisation of PH-domain probes during phagocytosis. (A) Time series showing phagocytosis of yeast cells (marked with asterisks) by Dictyostelium amoebae expressing various PH-domain-GFP fusion proteins as indicated. The phosphoinositide binding specificity of the PH domains is indicated. Bars, 5 µm. See also supplementary material Fig. S1a-f. (B) Phagocytosis of 1 µm beads by cells expressing GRP1 and TAPP1-PH-GFP. The brightfield images at t=0 seconds show the localisation of the beads before ingestion, the position of the beads during internalisation is marked by arrows.

View larger version (65K): [in a new window]   Fig. 4. Quantitative analysis of membrane localisation during phagocytosis. Fluorescence was measured at the plasma membrane (p) at the phagocytic cup/phagosome as well as in the adjacent cytoplasm (c), as indicated in inset in (A), and the ratio calculated as described in experimental procedures. Curves were aligned using `phagosome closure', the formation of a membrane enclosed phagosome as t=0 seconds. Data were averaged from 10 (GRP1), 13 (TAPP1), 7 (CRAC-PH), 9 (DAPP1-PH) and 12 (DAPP1G176A) cells.

View larger version (61K): [in a new window]   Fig. 5. Localisation of f-actin specific probes during phagocytosis. (A) Coronin-GFP localises to the site of particle engulfment, followed by a complex redistribution process (25-45 seconds) until the phagosome is propelled into the cytosol. Yeast marked by asterisks. Bar, 5 µm. See also supplementary material Fig. S2. (B) The f-actin specific probe ABD-GFP shows a similar localisation pattern during the uptake of a yeast cell (marked by asterisks). Bar, 5 µm. (C) Correlation of phagosome movement with changes in GRP1/TAPP1 binding to phagosomal membranes. The averaged fluorescence intensity data for GRP1 and TAPP1 are taken from Fig. 4. The movement of yeast particles during engulfment was tracked and the average velocity from five phagocytosis events from different cells plotted. Phagosome closure occurs at t=0 seconds.

View larger version (60K): [in a new window]   Fig. 6. Phagocytosis is impaired in PTEN null cells. (A) The uptake of TRITC-labelled yeast is reduced in PTEN null cells compared with wild-type AX3 cells. Low concentrations of the PI3-kinase inhibitor LY294002 slightly increase yeast uptake while high concentrations reduce phagocytosis of yeast particles. Average and standard deviation of four independent experiments. (B) Localisation of PTEN-GFP during phagocytosis of a yeast cell (asterisks). PTEN-GFP is expressed in PTEN null cells. See also supplementary material Fig. S3. (C) Comparison of GRP1-PH and TAPP1-PH membrane localisation during yeast phagocytosis in wild-type AX2 and PTENnull cells. Curves were aligned with phagosome closure occurring at t=0 seconds (PTENnull/GRP1: four cells averaged; PTENnull/TAPP1: 18 cells).

View larger version (64K): [in a new window]   Fig. 7. Cellular localisation of PtdIns(4)P during phagocytosis. The PtdIns(4)P-specific FAPP1-PH-GFP does not localise to the plasma membrane or phagosomal membrane during the uptake of yeast cells (marked with asterisk) (n: nucleus). Bar, 5 µm. See also supplementary material Fig. S4.

View larger version (88K): [in a new window]   Fig. 8. Changes in 3-phosphoinositide levels during macropinocytosis. Comparison of fluid-phase uptake during macropinocytosis using different PH-GFP fusion proteins. Arrows mark the position of the endocytic cup (0 seconds) and the forming macropinosomes. See also supplementary material Fig. S5.

View larger version (107K): [in a new window]   Fig. 9. Effect of cAMP stimulation on PH-domain localisation. (A) Snapshots of aggregation competent cells just before and after cAMP stimulation (final concentration 5 µM). Only PtdIns(3,4,5)P3 binding PH domains show plasma membrane translocation. 20 to 40 cells were recorded for each cell line. (B) Kinetics of membrane translocation of the PtdIns(3,4,5)P3-specific probes. Data averaged from eight (CRAC) to ten cells (DAPP1/GRP1). (C) Membrane localisation of GRP1-PH and CRAC-PH during chemotaxis. Arrows indicate the direction of the cAMP source. (D) Changes in GRP1-PH-GFP localisation following repositioning of a cAMP-filled micropipette (position marked by arrows). See also supplementary material Fig. S6.

View larger version (137K): [in a new window]   Fig. 10. 3-Phosphoinositide distribution during aggregation of Dictyostelium cells. (A) GRP1-PH-GFP localisation in aggregation stream cells moving towards a cAMP signalling centre (arrow indicates direction of movement). See also supplementary material Fig. S7a. The white square marks the position of the cells shown in (B). (B) Pseudocolour representation of the cell from (A) showing small transient changes in membrane binding (red: high fluorescence intensity; blue: low intensity). (C) Periodic changes in fluorescence intensity as measured in the centre of the aggregation stream. (D) CRAC-PH membrane localisation is restricted to the front of the cells in aggregation streams. See also supplementary material Fig. S7b. (E) Close-up of the cells marked in (D) showing the changes in CRAC-PH localisation. (F) Measurement of the periodic changes in CRAC translocation in the aggregation stream. (G) Cells expressing DAPP1G176A-PH-GFP moving towards an aggregation centre on the right-hand side (indicated by large arrow). The small arrows mark the membrane localisation of the probe in cells that seem to engulf the cells in front. (H) Close-up of the cell marked in (G) showing the engulfment of another Dictyostelium cell (asterisk) accompanied by a transient rise of PtdIns(3,4)P2 levels at the phagosome. See also supplementary material Fig. S7c. Bars, 50 µm.

View larger version (126K): [in a new window]   Fig. 11. Localisation of GRP1-PH and CRAC-PH in slug cells. (A,B) Cells expressing GRP1-PH-GFP show uniform membrane localisation in prestalk cells in the slug tip and in the posterior prespore cells. Bars, 50 µm. (C,D) Comparison of GRP1-PH and CRAC-PH localisation in cells in the slug tip. CRAC-PH localises to the front of the cell while GRP1-PH remains uniformly distributed. Fluorescence along the membrane was measured for each time point (Dormann et al., 2002a) and visualised by mapping the fluorescence intensities onto a circle in polar plots. The data are arranged like tree rings, with time - indicated in minutes - progressing from the inner to the outer circle. To align the data for the time series the circles are plotted so that the rightmost part of the cell membrane lies at the 0° position as indicated by the asterisks. Fluorescence intensities are colour-coded: low intensity blue, high intensity red. The GRP1-PH polar plot shows a mostly uniform fluorescence distribution while the CRAC-PH plot reflects the polarised membrane localisation. Arrows indicate direction of cell movement. Bars, 10 µm. (E,F) Transfer of slugs to agar containing 250 µM of the PI3-kinase inhibitor LY294002 leads to rapid loss of GRP1-PH-GFP plasma membrane localisation; however, CRAC-PH-GFP often accumulates in bright cytoplasmic structures (arrows). Bars, 30 µm

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