|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 3. Re-expression of p190 Rho-GAP in gene-deficient cells restores the functional response to plexin activation. (A) Fibroblasts derived from wild-type or from p190–/– mouse embryos (Brouns et al., 2000) were engineered to express PLXNB1 (see B for protein expression analysis) and then treated with 10 mM Sema4D for 1 hour to test their collapse response (in analogy to Fig. 2B). Scale bar: 20 µm. (B) PLXNB1-expressing p190–/– knockout fibroblasts were engineered to express exogenous p190 or its inactive mutant p190RA (both conjugated with GFP). Protein expression levels were analysed by immunoblotting with specific antibodies. Hsp90 expression was determined as loading reference. (C) Merged fluorescence images of the cells described in B after treatment with 10 nM Sema4D for 30 minutes to induce cellular collapse: the red channel reveals PLXNB1, detected with specific antibodies (EC-6.9), while the green channel shows p190-GFP. Co-expression of PLXNB1 with p190, but not with its inactive mutant, is required and sufficient to rescue the functional response to Sema4D in these cells. The asterisk marks a cell expressing PLXNB1 but not p190, which is insensitive to the ligand. Scale bar: 20 µm. The fraction of GFP-positive collapsed cells in each condition was counted and it is shown at the bottom. (D) p190-deficient fibroblasts expressing PLXNB1, and further transfected with p190-GFP or its inactive mutant p190RA-GFP (the same shown in A), were grown on fibronectin-coated coverslips and treated with 5 nM Sema4D for 5 minutes. Cells were then fixed and focal adhesions revealed in the red channel with anti-paxillin antibodies. After this short stimulation, very few cells underwent collapse [consistent with that described in 3T3 fibroblasts (see Barberis et al., 2004)], however, focal adhesions were disassembled in most of the cells expressing functionally competent p190-RhoGAP (identified by GFP expression). In contrast, semaphorin stimulation had no effect in cells lacking p190 or expressing the mutant devoid of RhoGAP activity (p190RA). Scale bar: 20 µm. The fraction of GFP-positive cells containing focal adhesion was counted and it is shown at the bottom. (E) Affinity purification of RhoA-GTP (by rhotekin-RBD pull-down) from equal amounts of protein lysates of p190–/– fibroblasts expressing PLXNB1 and the indicated exogenous p190 proteins, after treatment with 10 nM Sema4D for 15 minutes. Plexin-mediated Rho inactivation is abrogated in the absence of p190, but it is rescued by expression of exogenous p190-GFP in the gene-deficient cells. The mutated form of p190 (p190RA), lacking GAP activity, is unable to rescue the function. The effect was quantified by measuring the relative amounts of active RhoA in each condition (band intensity of RhoA-GTP versus total RhoA) and it is shown at the bottom.
|
