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First published online May 10, 2006
doi: 10.1242/10.1242/jcs.02912

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Elimination of plasma membrane phosphatidylinositol (4,5)-bisphosphate is required for exocytosis from mast cells

Gerald R. V. Hammond^1,*,, Stephen K. Dove², Alastair Nicol³, Jef A. Pinxteren⁴, Daniel Zicha³ and Giampietro Schiavo^1,

¹ Molecular Neuropathobiology, Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, London WC2A 3PX, UK
² School of Bioscience, University of Birmingham, Birmingham, B15 2TT, UK
³ Light Microscopy Laboratories, Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, London WC2A 3PX, UK
⁴ Flanders Interuniversity Institute for Biotechnology, Department of Medical Protein Research, Albert Baertsoenkaai 3, 9000 Ghent, Belgium

View larger version (68K): [in a new window]   Fig. 1. PtdIns(4,5)P2 is enriched at the plasma membrane of resting RPMCs. (A) The monoclonal anti-PtdIns(4,5)P2 antibody 2C11 stains the plasma membrane. RPMCs were fixed and stained at 4°C with (a,b) 2C11 (red) and Draq5TM (blue) or (c) CM-DiI as described in Materials and Methods; (a) shows an equatorial confocal section, (b,c) are projections of confocal sections taken at 1 µm intervals throughout the cell. (B) 2C11 binds PtdIns(4,5)P2 and PtdIns(3,4,5)P3. 2C11 was pre-incubated with 1000-fold molar excess of the indicated inositol phosphate before staining as in (A); equatorial confocal sections are shown. (C) 2C11 specifically detects PtdIns(4,5)P2 on the plasma membrane of RPMCs. Cells were preincubated with 50 µM of the indicated PH domain before staining (b,c), or with 1 mM neomycin during staining with 2C11 (a); equatorial confocal sections are shown. (D) PtdIns(4,5)P2 colocalises with the cortical actin cytoskeleton. Cells were co-stained with 2C11 (red) and Alexa Fluor 488-phalloidin (green) as described in Materials and Methods. A projection of confocal sections taken at 1 µm intervals throughout the cell is shown. (A-C) Micrographs are at the same magnification, except (Ac), which is at the same magnification as (D). Bars, 10 µm.

View larger version (55K): [in a new window]   Fig. 2. Plasma membrane PtdIns(4,5)P2 is transiently depleted during degranulation. (A) Cells were fixed with ice-cold 3% glutaraldehyde and stained either at rest or after stimulation for the indicated times with compound 48/80. First and third row of panels show merged images of 2C11 (red) and Alexa Fluor 647-concanavalin A (blue). Second and fourth row of panels show fluorescence intensity profiles for 2C11. Bar, 10 µm. Ctrl indicates that 2C11 has been omitted during staining. *, contaminating neutrophil. (B,C) 2C11 fluorescence was quantified as described in Materials and Methods. (B,C) Mean fluorescence (± s.e.m.) normalised to the resting level (B), distribution of fluorescence at the indicated time points (C); n>100 cells per time point. (D) RPMCs were loaded with unlabelled or [2-3H]inositol, and stimulated for the indicated time period with compound 48/80. Cells were either fixed and stained with 2C11 and quantified as in B (2C11), or extracted and analysed for PtdIns(4,5)P2 content by HPLC ([2-3H]-PtdIns(4,5)P2).

View larger version (36K): [in a new window]   Fig. 3. Phospholipase C causes the depletion of plasma membrane PtdIns(4,5)P2, and the release of Ca2+ from intracellular stores. (A,B) PLC inhibitors prevent mast cell degranulation. RPMCs were pre-incubated with U73-122 and/or U73-343 for 5 minutes (A), or Et-18-OMe (or EtOH, as vehicle control) for 20 minutes (B), at the indicated concentration before stimulation with 48/80 at 25°C. After 10 minutes, the medium was assayed for released ß-hexosaminidase (ß-hex) activity. Data are means of triplicates ± s.e.m. (C) PLC inhibitors block depletion of PtdIns(4,5)P2. Mast cells were untreated (ctrl) or incubated with 5 µM BAPTA/AM or as described in A and B. Subsequently, degranulation was evoked with 48/80 for 30 seconds. Cells were fixed and stained with 2C11 (red) and Alexa Fluor 647-concanavalin A (blue). Bar, 10 µm. (D) PLC inhibitors block Ca2+ signalling. RPMCs were loaded for 20 minutes with Fluo3/AM, pre-incubated with the indicated compound as in A-C or for 20 minutes with 5 µM BAPTA/AM, and activated with 48/80. Normalised fluorescence intensity traces are shown from a single representative cell for each condition.

View larger version (34K): [in a new window]   Fig. 4. PLC causes depletion of PtdIns(4,5)P2 from permeabilised mast cells. (A) RPMCs were permeabilised with SL-O in the presence of 100 µM MgATP and 3 mM Ca:EGTA at pCa 7, or 100 µM MgATP, 10 µM GTPS and 3 mM Ca:EGTA at pCa 5 for 3 minutes at 30°C. Cells were then fixed and stained with 2C11 (red) and Alexa Fluor 647-concanavalin A (blue). Bar, 10 µm. (B-D) PLC inhibitors prevent PtdIns(4,5)P2 depletion and block degranulation of permeabilised cells. Mast cells were permeabilised as described in (A) at pCa 5, 100 µM MgATP and 10 µM GTPS, in the presence of U73-122 or U73-343 (B), neomycin (C), 40 µM Et-18-OMe, 100 µM LY294002 or LY303511, and 5 mM ß-GP (D) as indicated. (E) RPMCs were permeabilised in the presence of 100 µM MgATP and 300 µM Ca:EGTA at pCa 8 at 30°C. After 2 minutes, the indicated peptide was added to 100 µM. After a further 5 minutes, cells were activated by the addition of the indicated buffers at the same concentrations as (A). The numbers of degranulated cells were counted, and the numbers normalised to the control value for each experiment. Values represent the means of three or more independent experiments ± s.e.m., with the exceptions of U73-343 and LY303511, which are means ± range of duplicate experiments. 0.4% EtOH is the vehicle control for 40 µM Et-18-OMe.

View larger version (55K): [in a new window]   Fig. 5. (A) Mast cells were permeabilised with SL-O in the presence of 100 µM MgATP, 10 µM GTPS and 3 mM Ca:EGTA at pCa 5 for 3 minutes at 30°C as in Fig. 4A, in the presence of 10 µM U73-122 or U73-343, 3 mM neomycin, 40 µM Et-18-OMe, 100 µM LY294002, 100 µM LY303511 or 5 mM ß-GP. (B) Cells were permeabilised in the presence of 300 µM Ca:EGTA at pCa 8; after two minutes, the indicated peptide was added to 100 µM. After a further 5 minutes at 30°C, Ca:EGTA at pCa 5 and GTPS were added to the same final concentrations as (A), and the cells incubated for 5 minutes. Cells were then fixed and stained with 2C11 (red) and Alexa Fluor 647-concanavalin A (blue). Top panels show merged images of 2C11 and concanavalin A; bottom panels show the fluorescence intensity profile for 2C11. Bar, 10 µm.

View larger version (34K): [in a new window]   Fig. 6. SigD depletes plasma membrane PtdIns(4,5)P2 and inhibits mast cell degranulation. (A) RPMCs were permeabilised with SL-O in the presence of 100 µM MgATP and 300 µM Ca:EGTA at pCa 8 in the absence (Ctrl) or presence of 100 µg/ml of SigD wild-type or the inactive SigDC462S mutant. After 10 minutes at 30°C, cells were fixed and stained with 2C11 and Alexa Fluor 488-phalloidin. Top panels show phalloidin staining, bottom panels show the fluorescence intensity profile for 2C11. Bar, 10 µm. (B) Mast cells in suspension were treated as in A. After 10 minutes at 30°C, cells were kept inactive by addition of 3 mM Ca:EGTA at pCa 7, or stimulated with 3 mM Ca:EGTA at pCa 5 and 100 µM GTPS (SigDC462S) as indicated. After a further 10 minutes, cells were quenched with ice-cold 5 mM EGTA, centrifuged and supernatants assayed for ß-hexosaminidase (ß-hex) activity.

View larger version (23K): [in a new window]   Fig. 7. DAG partially restores exocytosis from permeabilised mast cells. RPMCs in suspension were permeabilised for the indicated time at pCa 8 and then stimulated as described in Fig. 6B. Unstimulated cells (pCa 7) released 2% of their total ß-hexosaminidase (ß-hex) activity. (A) Cells were permeabilised for 10 minutes in the absence (Ctrl) or presence of 0.3 U/ml PtdIns-PLC, with or without 20 µM Et-18-OMe as indicated. (B) In vitro activity of PtdIns-PLC against [2-3H]-PtdIns in the absence or presence of 20 µM Et-18-OMe. Boiled PtdIns-PLC refers to 0.3 U/ml of the enzyme inactivated at 110°C for 15 minutes. (C) Indicated DAG analogues were applied for 10 minutes before stimulation. (D) Combined effect of PtdIns-PLC and SigD. Mast cells were permeabilised in the presence of 0.3 U/ml PtdIns-PLC and 100 µg/ml SigD for 10 minutes as indicated; refers to the difference in ß-hex release between incubations with and without SigD. (E) Effect of PtdIns-PLC on the IC50 of neomycin. Cells were permeabilised in the presence of the indicated concentration of neomycin with or without 0.3 U/ml PtdIns-PLC for 5 minutes before stimulation. Data are normalised to the levels of secretion observed without neomycin, which were 92±4% of the total cellular ß-hex activity in the presence of PtdIns-PLC, and 72±4% in its absence. Data are means ± s.e.m. (n=3) for stimulated cells (for inactive cells at pCa 7, n=1).