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

JCS02432 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. RBL-2H3 cell viability is unaffected by maintenance in suspension. Cells were detached from substrate and maintained in suspension.  At each time point 100 ml of cell suspension was aliquoted into six wells of a 96-well plate.  These cells were allowed to adhere for 1 hour before the media containing any dead cells was removed.  Each well was washed in PBS to remove any further debris. The number of cells in each well was subsequently determined as described in Materials and Methods.  The results show that over time, up to 4.5 hours in suspension, the number of viable cells able to adhere to serum-coated plastic was similar to the number of viable cells able to adhere immediately following release from substrate (t=0).  Similar results were obtained using a Trypan Blue exclusion protocol (data not shown).

• Supplemental Figure 2 -

  Fig. S2. Inhibition of PLD decreases adhesion in various cell types. Cells were detached from substrate and treated with 0.5% butan-1-ol.  The ability of these cells to adhere to serum-coated plastic was quantified as described in Materials and Methods and here is expressed as a percentage of untreated cells adhered under the same conditions.  Leukocytes (RBL-2H3), fibroblasts (KS1.6), epithelial cells (HEK293), myoblasts (L6). In all cases inhibition of PLD reduced the level of adhesion indicating that the activation of this enzyme is important in the regulation of adhesion in many tissue types.  Using Student’s t test, P values of less than 0.02 were obtained for all cell types treated with butan-1-ol in comparison to their untreated counterparts indicating that this effect is statistically significant for each cell type.

• Supplemental Figure 3 -

  Fig. S3. Adhesion of RBL-2H3 cells to different substrates. Cells were detached from substrate and their ability to adhere to serum-coated, FN-coated or ICAM1-coated plastic following treatment with vehicle, PtdOH or PtdIns(4,5)P₂ was quantified as described in Materials and Methods. Results are expressed here as a percentage of untreated cells adhered to serum-coated plastic. The results show that in the absence of exogenously added lipid RBL-2H3 cells were able to adhere to each well of serum, FN and ICAM1 and in all subsequent experiments the amount of FN or ICAM1 in each well was titrated such that the total level of adhesion to each of these surfaces was equivalent to the maximum achieved to serum alone (data not shown).  Following a 60-minute incubation of cells in suspension, more than 90% of the PLD activity stimulated by the detachment from substrate had declined with a corresponding decrease in adhesion (Fig. 1). In these cells, as identified in Fig. 2, adhesion to serum-coated plastic of cells was re-stimulated following the addition of diC8-PtdOH or diC8-PtdIns(4,5)P₂. Furthermore, adhesion to FN was greater than to serum in the absence of PLD activity but was also stimulated by the addition of either cell-permeable PtdOH or PtdIns(4,5)P₂.  Therefore, whereas PtdOH and PtdIns(4,5)P₂ generation regulate some adhesion to FN, probably mechanisms other than those regulated by PLD also control adhesion to FN.  More in line with adhesion to serum than to FN, adhesion to ICAM1 was predominantly dependent upon PLD activity and could also be stimulated by cell-permeable PtdOH and PtdIns(4,5)P₂.

• Supplemental Figure 4 -

  Fig. S4.  Increasing expression levels of PLD2 stimulate adhesion more than the equivalent level of PLD1. Adhesion of RBL-2H3 cells transfected with PLD constructs was quantified following cellular detachment from substrate as detailed in Materials and Methods. Low, Medium and High correspond to arbitrary ranges of expression that were equally divided but not overlapping.  Expression ranges were determined by detection of GFP fluorescence on a FACs instrument. 

• Supplemental Figure 5A -

  Fig. S5A.  PLD2 colocalises with all splice variants of the PIPkin Ig family. RBL-2H3 cells were co-transfected with GFP-tagged PLD2 (green) and FLAG-tagged a, b or c splice variants of PIPkin Ig (red).  Cells were detached from substrate at least 16 hours post-transfection and fixed and stained as detailed in Materials and Methods.  No difference in the colocalisation between PLD2 and each splice variant was observed. Thus PLD2, but not PLD1, is found within a similar localisation to the PtdIns(4,5)P₂-generating enzyme subfamily PIPkin Ig at the plasma membrane following detachment of cells from substrate and during the initial stages of adhesion.

• Supplemental Figure 5B -

  Fig. S5B.  PLD2 but not PLD1 colocalises with the PLCd1 PH domain at the plasma membrane. RBL-2H3 cells were co-transfected with HA-tagged PLD1 or PLD2 (red) and the GFP-tagged PH domain from PLCd1 (green).  Cells were detached from substrate at least 16 hours post-transfection and fixed and stained as detailed in Materials and Methods. The PLCd1 PH domain specifically binds to PtdIns(4,5)P₂ thereby revealing the localisation this lipid within membranes.  The images here show that PLD2, but not PLD1, is found within a similar localisation to PtdIns(4,5)P₂ at the plasma membrane following detachment of cells from substrate and during the initial stages of adhesion.

• Supplemental Figure 6 -

  Fig. S6.  Transfection of PLD2 but not PLD1 induces more rapid spreading than non-transfected cells. Cells were transfected with GFP-tagged PLD1 or PLD2.  Cells were detached from substrate and immediately re-plated on to serum-coated glass slides. Slides were fixed after 10 or 30 minutes, permeabilised and stained with Alexa Fluor546-conjugated phalloidin to detect F-actin.  It can be seen that the majority of PLD1 remains within an intracellular localisation during the course of this experiment and has little effect upon the morphology of the cells in comparison to non-transfected cells in the same field. By contrast, at both 10 and 30 minutes the majority of PLD2 is co localised at the cell periphery with F-actin and the morphology of the transfected cells is vastly different to the non-transfected cells in the same field; these cells have a far more villous and convoluted cortical plasma membrane indicative of cellular spreading and at 30 minutes this can be clearly seen as the PLD2 transfected cells have a greatly increased cell ‘footprint’ area than PLD1 or non-transfected cells.

• Supplemental Figure 7A -

  Fig. S7A.  PLD is upstream of integrin activation in the regulation of adhesion. The requirement for intracellular signals to increase the binding affinity of cell surface integrins for their substrates can be bypassed following the addition of extracellular Mn²⁺ to directly activate the integrins.  Following treatment with 0.5% butan-1-ol–tert-butanol, the level of adhesion was quantified in the absence and presence of 10 mM Mn²⁺.  Results here suggest that integrin activation may be downstream of PLD activation as the retardation of adhesion brought about by inhibiting PLD signalling, can be overcome by the direct activation of integrins with Mn²⁺. Detachment from substrate may not optimally activate cell surface integrins as Mn²⁺ treatment mediates a greater level of adhesion.

• Supplemental Figure 7B -

  Fig. S7B.  PLD2 but not PLD1 colocalises with talin at the plasma membrane. RBL-2H3 cells were co-transfected with GFP-tagged PLD1 or PLD2 (green) and stained for endogenous talin (red). Cells were detached from substrate at least 16 hours post-transfection and fixed and stained as detailed in Materials and Methods.  The images here show that PLD2 but not PLD1 is found within a similar localisation to talin at the plasma membrane following detachment of cells from substrate and during the initial stages of adhesion.