FIG 7.
Spatial cross talk between K-Ras and H-Ras depends on PS availability in the PM. (a) PM sheets prepared from wild-type BHK cells (WT), wild-type BHK cells treated with 1 μM latrunculin for 5 min (LA), and Cav-1KD BHK cells (Cav-1KD) expressing both GFP–H-RasG12V and RFP–K-RasG12V were labeled with 2-nm anti-RFP and 6-nm anti-GFP gold particles. Heterotypic coclustering between H-Ras and K-Ras was quantified using bivariate K functions, summarized as mean LBI values ± SEM. Similar experiments were conducted on cells coexpressing GFP-LactC2 and RFP–H-RasG12V, and the extent of coclustering of PS and H-RasG12V was quantified using bivariate K functions summarized as mean LBI values ± SEM. Statistical analysis was performed using Mann-Whitney tests (*, P < 0.05). (b) After treatment with 1 μM latrunculin for 5 min (LA), PM sheets were prepared from BHK cells expressing GFP–K-RasG12V alone (control), GFP–K-RasG12V and RFP–H-RasG12V (+HG12V), or GFP–K-RasG12V and RFP-CTH (+CTH). The sheets were immunogold labeled, and after EM imaging, the extent of GFP–K-Ras clustering was quantified using univariate K functions, shown as mean Lmax values ± SEM. Similar experiments were conducted in Cav1-KD cells. (c) BHK cells expressing GFP–K-RasG12V alone (white bar) and BHK cells or Cav-1KD cells coexpressing GFP–K-RasG12V and RFP–H-RasG12V (blue bars) were analyzed in FLIM experiments. Where indicated, measurements were also made on BHK cells treated with 1 μM latrunculin for 5 min (+LA). For a control, reference homotypic K-Ras clustering was examined in BHK cells coexpressing GFP–K-RasG12V and RFP–K-RasG12V (yellow bar). Each GFP fluorescence lifetime value is averaged for a single cell. Data were collected from multiple cells and are shown as means ± SEM (n ≥ 60 cells from 3 independent experiments). Statistical significance was evaluated using one-way ANOVA (*, P < 0.001). (d) Representative FLIM images of the BHK cells analyzed in panel c.