1. Volume 6, Issue 5, Pages 588-597 (May 1996)
Stimulation of actin stress fibre formation mediated by activation of phospholipase D 
Michael J. Cross, Sally Roberts, Anne J. Ridley, Matthew N. Hodgkin, Allison Stewart, Lena Claesson- Welsh, Michael J.O. Wakelam 
Current Biology 
Volume 6, Issue 5, Pages (May 1996)
DOI: /S (02)

2. Figure 1
Stimulation of PLD activity by LPA in PAE cells. [3H]palmitate-labelled cells, preincubated with 30 mM butan-1-ol for 5 min, were stimulated with 10 μM LPA for increasing times and the generation of [3H]phosphatidylbutanol determined. Results are expressed as radioactivity in [3H]phosphatidylbutanol (mean d.p.m. ± S.D., n = 3) from a single experiment representative of two.
Current Biology 1996 6, DOI: ( /S (02) )

3. Figure 2
LPA stimulates PA and DAG generation in PAE cells. (a) Time course of LPA-stimulated PA generation. Cells were stimulated for the times indicated with 10 μM LPA. Results are expressed as pmoles PA per nmole of total phospholipid (n = 2) from a single experiment representative of two. (b) Time course of LPA-stimulated DAG generation. Cells were stimulated for the times indicated with 10 μM LPA. Results are expressed as pmoles DAG per nmole of phospholipid (n = 2) from a single experiment representative of two.
Current Biology 1996 6, DOI: ( /S (02) )

4. Figure 2
LPA stimulates PA and DAG generation in PAE cells. (a) Time course of LPA-stimulated PA generation. Cells were stimulated for the times indicated with 10 μM LPA. Results are expressed as pmoles PA per nmole of total phospholipid (n = 2) from a single experiment representative of two. (b) Time course of LPA-stimulated DAG generation. Cells were stimulated for the times indicated with 10 μM LPA. Results are expressed as pmoles DAG per nmole of phospholipid (n = 2) from a single experiment representative of two.
Current Biology 1996 6, DOI: ( /S (02) )

5. Figure 3
Effect of inhibitors upon LPA-stimulated PLD activity and PA mass. (a) Effect of inhibitors upon LPA-stimulated PLD activity. [3H]palmitate-labelled cells were incubated with pertussis toxin (25 ng ml−1 for 18 h), genistein (100 μm for 30 min) or Ro (10 μM for 10 min) prior to the addition of 10 μM LPA in the presence of 30 mM butan-1-ol for 5 min. Results are expressed as radioactivity in [3H]phosphatidylbutanol (mean d.p.m. ± S.D., n = 3) from a single experiment representative of three. (b) Effect of PA and DOG on PLD activity. [3H]palmitate-labelled cells were incubated for 1 min with 10 mM butan-1-ol prior to stimulation for 5 min with 3 μM LPA, PA or DOG. Results are expressed as radioactivity in [3H]phosphatidylbutanol (mean d.p.m. ± S.D., n = 3) from a single experiment representative of two. (c) Effect of modulators of LPA-stimulated PLD activity on PA generation. Cells were incubated with either pertussis toxin (25 ng ml−1 for 18 h), genistein (100 μm for 30 min), butan-1-ol (10 mM for 1 min) or butan-2-ol (10 mM for 1 min), prior to stimulation with 3 μM LPA for 1 min. Results are expressed as PA generation (% increase over basal) from a single experiment (n = 3) representative of two. Mean basal PA concentration = 2.41 ± 0.3 pmoles PA per nmole total phospholipid. (d) Effect of alcohols on LPA-stimulated [3H]phosphatidylbutanol generation. [3H]palmitate-labelled cells were incubated for 1 min with 10 mM butan-1-ol, 10 mM butan-2-ol, or 10 mM butan-1-ol plus 10 mM butan-2-ol, prior to the addition of 3 μM LPA for 5 min. Results are expressed as radioactivity in [3H]phosphatidylbutanol (mean d.p.m. ± S.D., n = 3) from a single experiment representative of two.
Current Biology 1996 6, DOI: ( /S (02) )

6. Figure 3
Effect of inhibitors upon LPA-stimulated PLD activity and PA mass. (a) Effect of inhibitors upon LPA-stimulated PLD activity. [3H]palmitate-labelled cells were incubated with pertussis toxin (25 ng ml−1 for 18 h), genistein (100 μm for 30 min) or Ro (10 μM for 10 min) prior to the addition of 10 μM LPA in the presence of 30 mM butan-1-ol for 5 min. Results are expressed as radioactivity in [3H]phosphatidylbutanol (mean d.p.m. ± S.D., n = 3) from a single experiment representative of three. (b) Effect of PA and DOG on PLD activity. [3H]palmitate-labelled cells were incubated for 1 min with 10 mM butan-1-ol prior to stimulation for 5 min with 3 μM LPA, PA or DOG. Results are expressed as radioactivity in [3H]phosphatidylbutanol (mean d.p.m. ± S.D., n = 3) from a single experiment representative of two. (c) Effect of modulators of LPA-stimulated PLD activity on PA generation. Cells were incubated with either pertussis toxin (25 ng ml−1 for 18 h), genistein (100 μm for 30 min), butan-1-ol (10 mM for 1 min) or butan-2-ol (10 mM for 1 min), prior to stimulation with 3 μM LPA for 1 min. Results are expressed as PA generation (% increase over basal) from a single experiment (n = 3) representative of two. Mean basal PA concentration = 2.41 ± 0.3 pmoles PA per nmole total phospholipid. (d) Effect of alcohols on LPA-stimulated [3H]phosphatidylbutanol generation. [3H]palmitate-labelled cells were incubated for 1 min with 10 mM butan-1-ol, 10 mM butan-2-ol, or 10 mM butan-1-ol plus 10 mM butan-2-ol, prior to the addition of 3 μM LPA for 5 min. Results are expressed as radioactivity in [3H]phosphatidylbutanol (mean d.p.m. ± S.D., n = 3) from a single experiment representative of two.
Current Biology 1996 6, DOI: ( /S (02) )

7. Figure 3
Effect of inhibitors upon LPA-stimulated PLD activity and PA mass. (a) Effect of inhibitors upon LPA-stimulated PLD activity. [3H]palmitate-labelled cells were incubated with pertussis toxin (25 ng ml−1 for 18 h), genistein (100 μm for 30 min) or Ro (10 μM for 10 min) prior to the addition of 10 μM LPA in the presence of 30 mM butan-1-ol for 5 min. Results are expressed as radioactivity in [3H]phosphatidylbutanol (mean d.p.m. ± S.D., n = 3) from a single experiment representative of three. (b) Effect of PA and DOG on PLD activity. [3H]palmitate-labelled cells were incubated for 1 min with 10 mM butan-1-ol prior to stimulation for 5 min with 3 μM LPA, PA or DOG. Results are expressed as radioactivity in [3H]phosphatidylbutanol (mean d.p.m. ± S.D., n = 3) from a single experiment representative of two. (c) Effect of modulators of LPA-stimulated PLD activity on PA generation. Cells were incubated with either pertussis toxin (25 ng ml−1 for 18 h), genistein (100 μm for 30 min), butan-1-ol (10 mM for 1 min) or butan-2-ol (10 mM for 1 min), prior to stimulation with 3 μM LPA for 1 min. Results are expressed as PA generation (% increase over basal) from a single experiment (n = 3) representative of two. Mean basal PA concentration = 2.41 ± 0.3 pmoles PA per nmole total phospholipid. (d) Effect of alcohols on LPA-stimulated [3H]phosphatidylbutanol generation. [3H]palmitate-labelled cells were incubated for 1 min with 10 mM butan-1-ol, 10 mM butan-2-ol, or 10 mM butan-1-ol plus 10 mM butan-2-ol, prior to the addition of 3 μM LPA for 5 min. Results are expressed as radioactivity in [3H]phosphatidylbutanol (mean d.p.m. ± S.D., n = 3) from a single experiment representative of two.
Current Biology 1996 6, DOI: ( /S (02) )

8. Figure 3
Effect of inhibitors upon LPA-stimulated PLD activity and PA mass. (a) Effect of inhibitors upon LPA-stimulated PLD activity. [3H]palmitate-labelled cells were incubated with pertussis toxin (25 ng ml−1 for 18 h), genistein (100 μm for 30 min) or Ro (10 μM for 10 min) prior to the addition of 10 μM LPA in the presence of 30 mM butan-1-ol for 5 min. Results are expressed as radioactivity in [3H]phosphatidylbutanol (mean d.p.m. ± S.D., n = 3) from a single experiment representative of three. (b) Effect of PA and DOG on PLD activity. [3H]palmitate-labelled cells were incubated for 1 min with 10 mM butan-1-ol prior to stimulation for 5 min with 3 μM LPA, PA or DOG. Results are expressed as radioactivity in [3H]phosphatidylbutanol (mean d.p.m. ± S.D., n = 3) from a single experiment representative of two. (c) Effect of modulators of LPA-stimulated PLD activity on PA generation. Cells were incubated with either pertussis toxin (25 ng ml−1 for 18 h), genistein (100 μm for 30 min), butan-1-ol (10 mM for 1 min) or butan-2-ol (10 mM for 1 min), prior to stimulation with 3 μM LPA for 1 min. Results are expressed as PA generation (% increase over basal) from a single experiment (n = 3) representative of two. Mean basal PA concentration = 2.41 ± 0.3 pmoles PA per nmole total phospholipid. (d) Effect of alcohols on LPA-stimulated [3H]phosphatidylbutanol generation. [3H]palmitate-labelled cells were incubated for 1 min with 10 mM butan-1-ol, 10 mM butan-2-ol, or 10 mM butan-1-ol plus 10 mM butan-2-ol, prior to the addition of 3 μM LPA for 5 min. Results are expressed as radioactivity in [3H]phosphatidylbutanol (mean d.p.m. ± S.D., n = 3) from a single experiment representative of two.
Current Biology 1996 6, DOI: ( /S (02) )

9. Figure 4
Effect of LPA on actin reorganization. PAE cells were stimulated with 10 μM LPA for the times indicated, fixed and stained with rhodamine-conjugated phalloidin. (a,c,d,f,g) Bar, shown in (g), represents 30 μm. (b,e) Cells treated with 10 μM LPA for the times indicated, and photographed at higher magnification; bar represents 40 μm.
Current Biology 1996 6, DOI: ( /S (02) )

10. Figure 4
Effect of LPA on actin reorganization. PAE cells were stimulated with 10 μM LPA for the times indicated, fixed and stained with rhodamine-conjugated phalloidin. (a,c,d,f,g) Bar, shown in (g), represents 30 μm. (b,e) Cells treated with 10 μM LPA for the times indicated, and photographed at higher magnification; bar represents 40 μm.
Current Biology 1996 6, DOI: ( /S (02) )

11. Figure 4
Effect of LPA on actin reorganization. PAE cells were stimulated with 10 μM LPA for the times indicated, fixed and stained with rhodamine-conjugated phalloidin. (a,c,d,f,g) Bar, shown in (g), represents 30 μm. (b,e) Cells treated with 10 μM LPA for the times indicated, and photographed at higher magnification; bar represents 40 μm.
Current Biology 1996 6, DOI: ( /S (02) )

12. Figure 4
Effect of LPA on actin reorganization. PAE cells were stimulated with 10 μM LPA for the times indicated, fixed and stained with rhodamine-conjugated phalloidin. (a,c,d,f,g) Bar, shown in (g), represents 30 μm. (b,e) Cells treated with 10 μM LPA for the times indicated, and photographed at higher magnification; bar represents 40 μm.
Current Biology 1996 6, DOI: ( /S (02) )

13. Figure 4
Effect of LPA on actin reorganization. PAE cells were stimulated with 10 μM LPA for the times indicated, fixed and stained with rhodamine-conjugated phalloidin. (a,c,d,f,g) Bar, shown in (g), represents 30 μm. (b,e) Cells treated with 10 μM LPA for the times indicated, and photographed at higher magnification; bar represents 40 μm.
Current Biology 1996 6, DOI: ( /S (02) )

14. Figure 4
Effect of LPA on actin reorganization. PAE cells were stimulated with 10 μM LPA for the times indicated, fixed and stained with rhodamine-conjugated phalloidin. (a,c,d,f,g) Bar, shown in (g), represents 30 μm. (b,e) Cells treated with 10 μM LPA for the times indicated, and photographed at higher magnification; bar represents 40 μm.
Current Biology 1996 6, DOI: ( /S (02) )

15. Figure 4
Effect of LPA on actin reorganization. PAE cells were stimulated with 10 μM LPA for the times indicated, fixed and stained with rhodamine-conjugated phalloidin. (a,c,d,f,g) Bar, shown in (g), represents 30 μm. (b,e) Cells treated with 10 μM LPA for the times indicated, and photographed at higher magnification; bar represents 40 μm.
Current Biology 1996 6, DOI: ( /S (02) )

16. Figure 5
Effect of modulators of LPA-stimulated PA mass on stress fibre formation in PAE cells. (a) Control cells. (c,e,g) Cells were incubated for 1 min with either (c) 10 mM butan-1-ol or (e) 10 mM butan-2-ol, or (g) for 30 min with 100 μM genistein. (b,d,f,h) Cells shown in (a,c,e,g) after the addition of 3 μM LPA for 1 min. Cells were stained for actin filaments with rhodamine-conjugated phalloidin. Bar represents 30 μm.
Current Biology 1996 6, DOI: ( /S (02) )

17. Figure 5
Effect of modulators of LPA-stimulated PA mass on stress fibre formation in PAE cells. (a) Control cells. (c,e,g) Cells were incubated for 1 min with either (c) 10 mM butan-1-ol or (e) 10 mM butan-2-ol, or (g) for 30 min with 100 μM genistein. (b,d,f,h) Cells shown in (a,c,e,g) after the addition of 3 μM LPA for 1 min. Cells were stained for actin filaments with rhodamine-conjugated phalloidin. Bar represents 30 μm.
Current Biology 1996 6, DOI: ( /S (02) )

18. Figure 5
Effect of modulators of LPA-stimulated PA mass on stress fibre formation in PAE cells. (a) Control cells. (c,e,g) Cells were incubated for 1 min with either (c) 10 mM butan-1-ol or (e) 10 mM butan-2-ol, or (g) for 30 min with 100 μM genistein. (b,d,f,h) Cells shown in (a,c,e,g) after the addition of 3 μM LPA for 1 min. Cells were stained for actin filaments with rhodamine-conjugated phalloidin. Bar represents 30 μm.
Current Biology 1996 6, DOI: ( /S (02) )

19. Figure 5
Effect of modulators of LPA-stimulated PA mass on stress fibre formation in PAE cells. (a) Control cells. (c,e,g) Cells were incubated for 1 min with either (c) 10 mM butan-1-ol or (e) 10 mM butan-2-ol, or (g) for 30 min with 100 μM genistein. (b,d,f,h) Cells shown in (a,c,e,g) after the addition of 3 μM LPA for 1 min. Cells were stained for actin filaments with rhodamine-conjugated phalloidin. Bar represents 30 μm.
Current Biology 1996 6, DOI: ( /S (02) )

20. Figure 5
Effect of modulators of LPA-stimulated PA mass on stress fibre formation in PAE cells. (a) Control cells. (c,e,g) Cells were incubated for 1 min with either (c) 10 mM butan-1-ol or (e) 10 mM butan-2-ol, or (g) for 30 min with 100 μM genistein. (b,d,f,h) Cells shown in (a,c,e,g) after the addition of 3 μM LPA for 1 min. Cells were stained for actin filaments with rhodamine-conjugated phalloidin. Bar represents 30 μm.
Current Biology 1996 6, DOI: ( /S (02) )

21. Figure 5
Effect of modulators of LPA-stimulated PA mass on stress fibre formation in PAE cells. (a) Control cells. (c,e,g) Cells were incubated for 1 min with either (c) 10 mM butan-1-ol or (e) 10 mM butan-2-ol, or (g) for 30 min with 100 μM genistein. (b,d,f,h) Cells shown in (a,c,e,g) after the addition of 3 μM LPA for 1 min. Cells were stained for actin filaments with rhodamine-conjugated phalloidin. Bar represents 30 μm.
Current Biology 1996 6, DOI: ( /S (02) )

22. Figure 5
Effect of modulators of LPA-stimulated PA mass on stress fibre formation in PAE cells. (a) Control cells. (c,e,g) Cells were incubated for 1 min with either (c) 10 mM butan-1-ol or (e) 10 mM butan-2-ol, or (g) for 30 min with 100 μM genistein. (b,d,f,h) Cells shown in (a,c,e,g) after the addition of 3 μM LPA for 1 min. Cells were stained for actin filaments with rhodamine-conjugated phalloidin. Bar represents 30 μm.
Current Biology 1996 6, DOI: ( /S (02) )

23. Figure 5
Effect of modulators of LPA-stimulated PA mass on stress fibre formation in PAE cells. (a) Control cells. (c,e,g) Cells were incubated for 1 min with either (c) 10 mM butan-1-ol or (e) 10 mM butan-2-ol, or (g) for 30 min with 100 μM genistein. (b,d,f,h) Cells shown in (a,c,e,g) after the addition of 3 μM LPA for 1 min. Cells were stained for actin filaments with rhodamine-conjugated phalloidin. Bar represents 30 μm.
Current Biology 1996 6, DOI: ( /S (02) )

24. Figure 6
Effect of PA and DOG on stress fibre formation in PAE cells. (a) Control cells. (b,e) Cells were stimulated with (b) 3 μM PA or (e) 3 μM DOG for 5 min. (c,d) Cells were preincubated with (c) 10 mM butan-1-ol for 1 min, or with (d) 100 μM genistein for 30 min, before the addition of 3 μM PA for 5 min. Cells were stained for actin filaments with rhodamine-conjugated phalloidin. Bar represents 30 μm.
Current Biology 1996 6, DOI: ( /S (02) )

25. Figure 6
Effect of PA and DOG on stress fibre formation in PAE cells. (a) Control cells. (b,e) Cells were stimulated with (b) 3 μM PA or (e) 3 μM DOG for 5 min. (c,d) Cells were preincubated with (c) 10 mM butan-1-ol for 1 min, or with (d) 100 μM genistein for 30 min, before the addition of 3 μM PA for 5 min. Cells were stained for actin filaments with rhodamine-conjugated phalloidin. Bar represents 30 μm.
Current Biology 1996 6, DOI: ( /S (02) )

26. Figure 6
Effect of PA and DOG on stress fibre formation in PAE cells. (a) Control cells. (b,e) Cells were stimulated with (b) 3 μM PA or (e) 3 μM DOG for 5 min. (c,d) Cells were preincubated with (c) 10 mM butan-1-ol for 1 min, or with (d) 100 μM genistein for 30 min, before the addition of 3 μM PA for 5 min. Cells were stained for actin filaments with rhodamine-conjugated phalloidin. Bar represents 30 μm.
Current Biology 1996 6, DOI: ( /S (02) )

27. Figure 6
Effect of PA and DOG on stress fibre formation in PAE cells. (a) Control cells. (b,e) Cells were stimulated with (b) 3 μM PA or (e) 3 μM DOG for 5 min. (c,d) Cells were preincubated with (c) 10 mM butan-1-ol for 1 min, or with (d) 100 μM genistein for 30 min, before the addition of 3 μM PA for 5 min. Cells were stained for actin filaments with rhodamine-conjugated phalloidin. Bar represents 30 μm.
Current Biology 1996 6, DOI: ( /S (02) )

28. Figure 6
Effect of PA and DOG on stress fibre formation in PAE cells. (a) Control cells. (b,e) Cells were stimulated with (b) 3 μM PA or (e) 3 μM DOG for 5 min. (c,d) Cells were preincubated with (c) 10 mM butan-1-ol for 1 min, or with (d) 100 μM genistein for 30 min, before the addition of 3 μM PA for 5 min. Cells were stained for actin filaments with rhodamine-conjugated phalloidin. Bar represents 30 μm.
Current Biology 1996 6, DOI: ( /S (02) )

29. Figure 7
PA-  and LPA-induced stress fibre formation is inhibited by C3 transferase. Serum-starved PAE cells were microinjected with C3 transferase (8 μg ml−1) and the marker protein rat IgG (0.5 mg ml−1). Cells were either fixed after 20 min (a), or stimulated after 15 min with LPA (10 μM) for 2 min (b), or with PA (10 μM) for 5 min (c). Actin filaments were visualized with TRITC-labelled phalloidin. Arrows indicate injected cells, identified by staining with FITC-conjugated goat anti-rat IgG.
Current Biology 1996 6, DOI: ( /S (02) )

30. Figure 7
PA-  and LPA-induced stress fibre formation is inhibited by C3 transferase. Serum-starved PAE cells were microinjected with C3 transferase (8 μg ml−1) and the marker protein rat IgG (0.5 mg ml−1). Cells were either fixed after 20 min (a), or stimulated after 15 min with LPA (10 μM) for 2 min (b), or with PA (10 μM) for 5 min (c). Actin filaments were visualized with TRITC-labelled phalloidin. Arrows indicate injected cells, identified by staining with FITC-conjugated goat anti-rat IgG.
Current Biology 1996 6, DOI: ( /S (02) )

31. Figure 7
PA-  and LPA-induced stress fibre formation is inhibited by C3 transferase. Serum-starved PAE cells were microinjected with C3 transferase (8 μg ml−1) and the marker protein rat IgG (0.5 mg ml−1). Cells were either fixed after 20 min (a), or stimulated after 15 min with LPA (10 μM) for 2 min (b), or with PA (10 μM) for 5 min (c). Actin filaments were visualized with TRITC-labelled phalloidin. Arrows indicate injected cells, identified by staining with FITC-conjugated goat anti-rat IgG.
Current Biology 1996 6, DOI: ( /S (02) )

32. Figure 8
Schematic representation of the LPA-stimulated signalling cascade resulting in stress fibre formation in PAE cells.
Current Biology 1996 6, DOI: ( /S (02) )