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First published online June 23, 2005
doi: 10.1242/10.1242/jcs.02432

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Phospholipase D2 stimulates integrin-mediated adhesion via phosphatidylinositol 4-phosphate 5-kinase Ib

¹ CRUK Institute for Cancer Studies, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK
² Department of Pharmacology, University of Cambridge, Cambridge, CB2 1PD, UK

View larger version (27K): [in a new window]   Fig. 1. PLD activation regulates adhesion. (A) At each time point following detachment from substrate (t=0), the number of cells able to adhere to serum-coated plastic over a 5 minute period was measured. (B) At each time point following detachment from substrate (t=0), PtdBut accumulation was quantified over a 5 minute period as a measure of PLD activity in either cells allowed to immediately re-adhere (dotted line) or cells maintained in suspension (solid line). (C) The level of adhesion after 10 or 30 minutes on serum-coated plastic was quantified for cells that had previously been detached from substrate and treated for 5 minutes with 0.5% butan-1-ol/tert-butanol. Results are expressed as a percentage of the number of untreated cells adhered after 10 or 30 minutes. At 10 minutes, the level of untreated cells adhered were approximately 70% of total cells added to each well. At 30 minutes, the level of untreated cells adhered were approximately 90% of total cells added to each well (data not shown). Similar results were obtained in at least three separate experiments and error bars represent the standard deviation from the mean.

View larger version (28K): [in a new window]   Fig. 2. PtdIns(4,5)P2 regulates adhesion downstream of PLD activation. (A) Both in the absence or presence of 0.5% butan-1-ol and following treatment with either 100 µM PtdCho, 100 µM PtdOH, 50 µM DAG, 100 µM PtdIns(4,5)P2 or vehicle (control) the level of cellular adhesion to serum-coated plastic was quantified. Optimal concentrations of each lipid were determined in dose-response experiments. The dotted line represents the level of adhesion observed in control cells not treated with butan-1-ol. In the presence of butan-1-ol, treatment with lipid was considered to have completely recovered the loss of adhesion due to PLD inhibition if the level of adhesion was above this line (B) Adhesion to serum-coated plastic was quantified after cells had been maintained in suspension for the indicated time points and subsequently treated with PtdOH, PtdIns(4,5)P2 or vehicle (no lipid). Using a Student's t-test, P values of <0.05% were obtained (* and **). The level of adhesion for each individual lipid was obtained from an average of at least three experiments and error bars represent the standard deviation from the mean.

View larger version (42K): [in a new window]   Fig. 3. PtdOH and PtdIns(4,5)P2 regulate integrin-mediated adhesion. Following the maintenance of cells in suspension for 60 minutes to reduce PLD activity to basal levels, cells were treated with 10 mg ml–1 of function-blocking antibodies to either ß1 or ß2 integrins or vehicle (no antibody) and either 100 µM PtdOH, 100 µM PtdIns(4,5)P2 or vehicle (no lipid) before their adhesion was quantified to wells coated with (A) serum, (B) FN or (C) ICAM1. Using a Student's t-test, P values of <0.01% were obtained (*, ** and ***). Similar results were obtained in at least three separate experiments and error bars represent the standard deviation from the mean.

View larger version (24K): [in a new window]   Fig. 4. PLD2 stimulates adhesion. (A) Following transfection of GFP-tagged protein constructs, the level of adhesion to serum-coated plastic was measured. The level of adhesion for each individual transfectant was obtained from an average of three experiments and is expressed as a percentage difference to non-transfected cells. (B) The PtdBut accumulation over a 5-minute period was used as a measure of PLD activity of transfected cells following detachment from substrate. PLD1** corresponds to PLD1(H464D/K860E) and PLD2** corresponds to PLD2(H442D/K758E). (C) Following siRNA transfection, the levels of PLD1 mRNA was analysed by quantitative PCR. (D) Following siRNA transfection, the levels of PLD2 mRNA was analysed by quantitative PCR. (E) Following siRNA transfection, the level of PtdBut accumulation over a 5-minute period was quantified as a measure of PLD activity. (F) Following siRNA transfection, the level of adhesion to serum-coated plastic was quantified. Using a Student's t-test, P values of <0.001% were obtained (*) between these samples. Similar results were obtained in at least three separate experiments and error bars represent the standard deviation from the mean.

View larger version (23K): [in a new window]   Fig. 5. PIPkin Ib stimulates adhesion. (A) Following transfection with GFP-tagged PIPkin I and (and derivatives) and the GFP-tagged PH domain for PLC1, the level of adhesion to serum-coated plastic was quantified and is expressed as a percentage difference to non-transfected cells. Using a Student's t-test, a P value of <0.01% was obtained (*) between these transfectants. (B) The dose-response for increasing levels of PIPkin I transfection derived from the total levels observed in (A). `Low', `medium', `high' and `very high' correspond to arbitrary divisions in the level of expression. The levels of expression were determined by the quantity of GFP fluorescence detected by a FACs instrument. The level of adhesion for each individual transfectant was obtained from an average of three experiments and error bars represent the standard deviation from the mean. Using a Student's t-test, a P value of <0.01% was obtained (*) between these transfectants.

View larger version (37K): [in a new window]   Fig. 6. PLD2 regulates activation of PIPkin Ib. (A) The localisations of GFP-tagged PLD1 and 2 with FLAG-tagged PIPkin I were determined for cells in suspension (spun down on to a cover-slip). The data shown is that of PIPkin Ib, but Ia and Ic gave identical results (supplementary material Fig. S5A). The inset image represents an image at a further 10x magnification. (B) Following transfection with GFP, GFP-tagged PLD2, FLAG-tagged PIPkin IbD316K and co-transfection of GFP-tagged PLD2 and FLAG-tagged PIPkin IbD316K adhesion to serum-coated plastic was quantified. Using a Student's t-test, a P values of <0.05% was obtained (*) between these transfectants. (C) Following maintenance in suspension for 60 minutes, the level of adhesion to serum-coated plastic was quantified for cells transfected with GFP-tagged PIPkin Ib or the GFP-tagged PH domain from PLC1, in the presence of diC8-PtdOH or vehicle. The level of adhesion for each individual transfectant was obtained from an average of three experiments and error bars represent the standard deviation from the mean. Similar results were obtained when PLD-activity was suppressed in the presence of butan-1-ol (data not shown). (D) Following maintenance in suspension for 60 minutes, the localisation of GFP-tagged PLC1 was analysed in the presence of diC8-PtdOH or vehicle. Similar results were obtained when PLD-activity was suppressed in the presence of butan-1-ol (data not shown). All images are confocal and similar results were obtained in at least three separate experiments. Bar, 10 µm.