TGFβ-1 induces increase in Ras protein PM localization
To follow subcellular localization of H-ras and K-ras during TGFβ treatment we used breast cancer cell line MCF7 and transfected it with GFP tagged constitutively active GTP-bound H-ras G12V and K-ras G12V. The transfected cells were treated with TGFβ-1 for 2 days. The equal level of expression of fusion protein in samples with and without TGFβ-1 treatment was carefully checked and confirmed (Fig. S1a). The subcellular localization of fusion proteins was analyzed both qualitatively by visual inspection and quantitatively by calculating membrane-to-cytoplasm ratio (M-C ratio), when higher M-C ratio translated into higher accumulation of fusion protein on PM.
In case of H-ras a large proportion of cells showed cytoplasm localization in steady state and this proportion significantly decreased after TGFβ-1 treatment resulting in more than 91% of cells with H-ras on PM (Fig. 1a). In contrast, K-ras that showed a very similar proportion of cells with PM and cytoplasm localization in steady state as H-ras did not change this proportion even after TGFβ-1 addition. However, similar to H-ras, the total amount of K-ras bound to PM increased after TGFβ-1 addition (Fig. 1, Fig. S2).
To study the mechanism of increased membrane localization of H-ras in more detail we tested its truncated version without the catalytic domain (G-domain). It has been documented that the minimal membrane anchor part of H-ras (tH) requires presence of adjacent hypervariable linker region to be laterally segregated as H-ras G12V and K-ras G12V into cholesterol-independent microdomains where the signaling occurs 17,19. Therefore, we used CTH construct composed of both membrane anchor part and the hypervariable linker region tagged with CFP. Indeed, CTH followed the trend set by H-ras indicating that this membrane anchor part is sufficient for the protein response to TGFβ-1 treatment (Fig. 1, Fig. S2).
Our results thus show that TGFβ-1 triggers relocalization of H-ras from cytoplasm to PM and causes increased accumulation of K-ras in PM.
TGFβ-1 treatment triggers rise in positive membrane curvature
In a previous study we have proven on example of tN-ras, a minimal membrane anchor of the N-ras isoform, that Ras senses positive membrane curvature in in vitro reconstituted systems and that this membrane partitioning is essential for its enrichment in raft-like liquid ordered phases of membrane 13. In addition, H-ras and to a lesser extend also K-ras were shown to preferentially localize to positively curved membranes in vivo 14. Therefore we decided to investigate if changes in the partitioning of H-ras G12V, CTH and K-ras G12V during TGFβ-1 treatment are accompanied by the acquisition of positive membrane curvature. We transfected MCF7 cells with YFP tagged Nadrin N-BAR, a sensor of positive membrane curvature 20, and followed changes in Nadrin N-BAR localization triggered by TGFβ-1 (Fig. S1b).
At steady state Nadrin N-BAR localized almost entirely in cytoplasm, whereas several puncta of Nadrin N-BAR accumulated on the PM after treatment with TGFβ-1 indicating that the cells acquired increased positive membrane curvature (Fig. 2).
Apart from sensing positive membrane curvature the N-BAR domains were documented to bind negatively charged lipids. N-BAR domain of another N-BAR containing protein amphiphysin was shown to interact equally well with two lipids - PI(4,5)P2 and phosphatidylserine (PS) 21. To investigate the possible role of these protein-lipid interactions in observed responses of Nadrin N-BAR to TGFβ-1 we trasfected MCF7 cells with GFP-tagged PH domain of PLCdelta, sensor of PI(4,5)P2, 22 and C2 domain of Lactadherin, specifically binding PS 23(Fig. S1b).
In both cases the sensors of negatively charged lipids showed mostly PM localization that either remained unchanged (PLCdelta PH) or decreased (Lactadherin C2) after TGFβ-1 treatment. Similarly, the total amount of proteins on PM after TGFβ-1 treatment showed no significant difference in case of PLCdelta PH, but decreased significantly in case of Lactadherin C2 (Fig. 2). The effect observed for Lactadherin C2 is likely to be caused by redistribution of PS from PM because a proteomic study performed in MDKC cells did not detect any significant drop in total PS amount after EMT induction 24. It is also consistent with observation that PM localization of PS decreases with increasing positive membrane curvature whereas localization of PI(4,5)P2 is not significantly affected 14. Since the response of Nadrin N-BAR to TGFβ-1 treatment does not follow the trend observed in either PLCdelta PH or Lactadherin C2, we can therefore conclude that TGFβ-1 induced changes in Nadrin N-BAR PM localization are solely driven by changes in membrane curvature and indicate rise in positive membrane curvature.
Increased level of positive membrane curvature leads to elevated H-ras PM localization
To validate our theory that the increase in accumulation of Ras proteins in PM during TGFβ-1 treatment is driven by the rise of positive membrane curvature we performed a set of further experiments for which we used a fibroblast cell line NIH 3T3 as an independent system. NIH 3T3 cells were selected because fibroblasts are rich in caveolae, small PM invaginations about 60 nm in size, and thus contain high number of areas with positive membrane curvature 25. Indeed, both the M-C ratio and the proportion of cells with PM localization of Nadrin N-BAR were higher in NIH 3T3 cells than in MCF7 cells before or after TGFβ-1 treatment (Fig. S3). Similarly, both H-ras and CTH showed significantly higher association with PM in NIH 3T3 compared to situation in MCF 7 favoring the fact that H-ras and CTH also recognize positive membrane curvature (Fig. S3).
In contrast, recruitment of K-ras to PM decreased in NIH 3T3 compared to MCF7 cells (Fig. S3c). This result is consistent with previous observation in BHK cells where increase in positive membrane curvature also led to PM depletion of K-ras14. A possible explanation for this could lay in a different way how H-ras and K-ras associate with PM. PM localization of H-ras occurs through two lipid anchors - farnesyl and palmytoyl, whereas PM localization of K-ras, apart from its one lipid anchor (C-terminal farnesyl), largely depends on binding negatively charged lipids, especially phosphatidylserine (PS), via its polybasic domain (PBD) 26,27. Similar to K-ras, Lactadherin C2 showed also significantly reduced PM binding (Fig. S3e). Moreover, it was recently demonstrated that K-ras G12V strictly prefers PS with unsaturated acyl chains over fully saturated PS 28. Since caveolae are known to be composed mostly of lipids with saturated acyl chains, pool of unsaturated PS available for binding may be actually significantly smaller in caveolae-rich fibroblasts compared to epithelia cells and, in combination with overall lower PS level, it could represent a main limiting factor for K-ras G12V PM recruitment in NIH 3T3 cells.
To inspect if Ras proteins indeed preferentially localize to areas of high positive membrane curvature we co-transfected H-ras G12V with Nadrin N-BAR in NIH 3T3 cells and we observed a significant correlation in PM localization of both proteins (Fig. 3).
Disruption of membrane curvature causes drop in Ras proteins PM localization
The results of our experiments strongly suggests that recognition of positive membrane curvature is likely a driving mechanism behind increased PM targeting of Ras after TGFβ-1 treatment. Therefore, experiments reducing positive membrane curvature of cell membranes should cause release of Ras from PM.
One option how to reduce membrane curvature is to subject cells to hyposmotic shock. The cells start to swell as indicated by increased FM1-43 staining (Fig. 4, Fig. S4); 29 Moreover, osmotic swelling leads to rapid disappearance of caveolae 30. Drop in Nadrin N-BAR PM localization indeed confirmed that hyposmotic shock reduces positive membrane curvature in NIH 3T3 cells. H-ras and CTH were both rapidly released from PM following hyposmotic shock induction with response of CTH being more pronounced than the one of H-ras. K-ras also showed fast and stable release from plasma membrane. The drop in K-ras PM localization was much higher than in case of Lactadherin C2 indicating that reduction in PM PS level cannot be the only reason for K-ras release from PM (Fig. 4, Fig. S4). In contrast to Nadrin N-BAR that showed constant and gradual decrease in PM localization, the release of Ras isoforms from plasma membrane reaches sooner or later plateau reflecting possibly different ways of interaction with PM and membrane curvature recognition by these proteins (concave shape of N-BAR vs. lipid anchors of Ras) (Fig. S4).
Besides the hyposmotic shock that disturbs membrane curvature in general, positive membrane curvature can be specifically reduced by targeted lowering of the caveolae number. Expression of dominant negative caveolin (CavDGV) was shown to significantly reduce number of caveolae (reduction by 62% in BHK cells; 31. We performed co-expression of GFP-CavDGV with RFP-H-ras G12V in NIH 3T3 cells followed by quantitative analysis of H-ras PM localization. Indeed, we observed a decrease in H-ras PM localization when single (RFP-H-ras G12V) and double (GFP-CavDGV with RFP-H-ras G12V) transfected cells were compared (Fig. 5, Fig. S5). The reduction was partial because, consistently with previous observations, H-ras was detectable on PM even at high expression levels of CavDGV (Fig. 5, Fig. S5); 31. However, the effect of CavDGV expression on the PM localization of H-ras was even more evident from the negative correlation between the amount of H-ras associated with PM and the level of CavDGV expression (Fig. 5, Fig. S5).