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Molecular mechanisms regulating the subcellular localization of p95-APP1 between the endosomal recycling compartment and sites of actin organization at the cell surface

Cell Adhesion Unit, DIBIT, S. Raffaele Scientific Institute, Via Olgettina 58, 20132 Milano, Italy

View larger version (46K): [in a new window]   Fig. 1. (A) P95-APP1-derived constructs used in this study. Zn, zinc finger; GAP, ARF-GAP domain; ANK, ankyrin repeats; SHD, Spa2 homology 1 domain; COIL, coiled-coil region; PBS, paxillin binding subdomain; K39 indicates the amino acid substitution in the ARF-GAP domain of the p95-K39 construct. (B) Coimmunoprecipitation of p95-C2 with paxillin and PIX. Aliquots of a lysate from CEFs transfected with pFlag-p95-C2 (lanes 1-6) were immunoprecipitated with the mAb anti-Flag and blotted with anti-Flag to detect p95-C2 (lanes 1-3) or with anti-paxillin (lanes 4-6). Lysate from CEFs cotransfected with pFlag-p95-C2 and pXJ40-HA-ßPIX (lanes 7-9) were immunoprecipitated with the anti-Flag mAb. Immunoprecipitates were blotted with the anti-Flag mAb to detect p95-C2 (bottom panel) and with the anti-HA mAb to detect the coprecipitating ß-PIX polypeptide (top panel). Pre, beads from pre-clearing; IP, immunoprecipitates; nb, material not bound to beads. Molecular weight markers are indicated to the left of panels.

View larger version (116K): [in a new window]   Fig. 2. Subcellular localization of p95-C2, ß-PIX, PAK1 and p95-N4. (A-J) Localization of p95-C2, ß-PIX and PAK1 at large vesicles. Cells were cotransfected with pFlag-p95-C2 (A,C) and pCMV6m/Pak1 (B,D), or with pFlag-p95-C2 (F) and pXJ40-HA-ßPIX (G). (E) Cell transfected with CMV6m/Pak1 only. (H) Cell transfected with pXJ40-HA-ßPIX only. In (I,J) cells were cotransfected with pXJ40-HA-ßPIX (I) and pCMV6m/Pak1 (J). (K-O) Localization of p95-N4 at small vesicles. Cells were cotransfected with pFlag-p95-N4 (K) and pCMV6m/Pak1 (L), and with pFlag-p95-N4 (M) and pXJ40-HA-ßPIX (N). In (O), merged signals for p95-N4 (shown in red) and ß-PIX (shown in green) are shown. Cells were fixed one day after transfection and processed for indirect immunofluorescence as described in Materials and Methods. Bar, 10 µm.

View larger version (142K): [in a new window]   Fig. 3. Specific colocalization of p95-C2 with markers of the endocytic recycling compartment. In cells transfected with pFlag-p95-C2 the distribution of the p95-C2 polypeptide was compared with the distribution of the early endocytic marker EEA1 (A-E), with the markers for the recycling compartment Rab11 (F,G) and transferrin receptor (I,J), with the marker for late endosomal compartment LBPA (L-N), and with the lysosomal marker LEP100 (O-Q). H and K show the distribution in non-transfected cells of Rab11 and transferrin receptor, respectively. Cells were fixed and processed for indirect immunofluorescence one day after transfection. Bars, 10 µm. In (C), p95-C2 is shown in red, EEA1 is shown in green. In (N) and (Q), p95-C2 is shown in green, endocytic markers are shown in red.

View larger version (131K): [in a new window]   Fig. 4. Comparison of the distribution of p95-N4 with markers of the endocytic compartment. In cells transfected with pFlag-p95-N4 the distribution of the p95-N4 polypeptide was compared with the distribution of the early endocytic marker EEA1 (A-C), with the recycling endosomal compartment markers Rab11 (D-I) and transferrin receptor (J, K), with the marker for late endosomal compartment LBPA (L-N), and with the lysosomal marker LEP100 (O-Q). Bars, 10 µm (A-F,J-Q); 5 µm (G-I). In C, p95-N4 is shown in red and EEA1 is shown in green. In F and I, p95-N4 is shown in green, and Rab11 is shown in red. In N and Q, p95-N4 is shown in green, and endocytic markers are shown in red.

View larger version (89K): [in a new window]   Fig. 5. Comparison of the distribution of N27-Arf6 with that of distinct p95-APP1-derived polypeptides. Cells were cotransfected with pFlag-p95-N4 (A) and pcDNA-N27-ARF6 (B), with pFlag-p95-C2 (C,E) and pcDNA-N27-ARF6 (D,F). One day after transfection cells were fixed and processed for indirect immunofluorescence. In G the merged signals of p95-C2 (red) and N27-ARF6 (green) are shown. Bars, 10 µm.

View larger version (104K): [in a new window]   Fig. 6. Characterization of the effects of p95-C3 and p95-K39 expression in fibroblasts. (A) Cells transfected with pFlag-p95-C3 were stained for p95-C3 (a,c) and F-actin (b,d). The same cells are shown in a,b and in c,d, respectively. Cells cotransfected with pFlag-p95-C3 and pCMV6m/Pak1 (e,f), and with pFlag-p95-C3 and pXJ40-HA-ßPIX (g) were immunostained to detect p95-C3 (e), PAK1 (f) and ß-PIX (g). Colocalization of p95-C3 (h) with Rab11 (i). In (j), the distribution of p95-C3 (green) and that of EEA1 (red) are compared. Bars, 10 µm. (B) Identification of the ß-PIX/p95-K39/paxillin complex. Fibroblasts cotransfected with pFlag-p95-K39 and pXJ40-HA-ßPIX were lysed and incubated first with control Protein-A-Sepharose beads (left lane), and then with beads coated with the anti-Flag mAb (right lane). After SDS-PAGE and blotting, filters were incubated with anti-Flag mAb (top), anti-PIX pAb (middle), and anti-paxillin mAb (bottom). Molecular weight markers are indicated to the left of the blot. (C) Cells transfected with pFlag-p95-K39 were analyzed for the distribution of p95-K39 (a), and F-actin (b); c shows the localization ß-PIX at large vesicles in a cell cotransfected with pFlag-p95-K39 and pXJ40-HA-ßPIX; d shows the localization at large vesicles of PAK in a cell cotransfected with pFlag-p95-K39 and pCMV6m/Pak1. Bars, 10 µm.

View larger version (77K): [in a new window]   Fig. 7. Effects of p95-C, p95-N5, and p95-C5 on cell morphology. Fibroblasts transfected with pFlag-p95-C, pFlag-p95-N5, or pFlag-p95-C5 were used for immunofluorescence. (A,B) The effects of each construct on the protrusive activity of the transfected cells was quantified as described in Materials and Methods, and compared to the protrusive activity of non-transfected cells (Control). (A) Percentage of cells with protrusions. (B) Number of protrusions per cell. For each value, three independent experiments were examined; bars represent the s.e.m. (C) Cells were transfected with pFlag-p95-N5 (a,c,f,h,j), with pFlag-p95-C (b), or with pFlag-p95-C5 (l) and treated for immunofluorescence one day after transfection. The distribution of p95-N5 was compared with the distribution of endogenous Rab11 (d,i,k), and of endogenous EEA1 (g). In (e) the signal for p95-N5 is shown in green, Rab11 is shown in red. Bars, 10 µm (a-i,l,m); 5 µm (j,k). Arrows point to corresponding areas of the cytoplasm in j and k.

View larger version (132K): [in a new window]   Fig. 8. Distribution of paxillin in fibroblasts expressing distinct p95-APP1-derived polypeptides. Cells expressing the indicated constructs were treated for immunofluorescence one day after transfection. P95-APP1-derived constructs were detected with an anti-Flag antibody (A,C,E,G,I), ß-PIX was detected with the anti-PIX pAb (L), and paxillin was detected with an anti-paxillin mAb (B,D,F,H,J,K,M). In K, at the center of the field the distribution of paxillin in a cell transfected with p95-K39 is shown. The same field is shown in (A,B), (C,D), (E,F), (G,H), (I,J), and (L,M). Bars, 10 µm.

View larger version (125K): [in a new window]   Fig. 9. Relocalization of p95-C2, p95-C3, paxillin, PIX, and PAK to the cell periphery by activated Rac. Fibroblasts were transfected or cotransfected with the following plasmids: pFlag-p95-C2 (A); pFlag-p95-C2 and pFlag-V12-Rac1B (B,C,E,L,M,P,Q); pFlag-p95-C2 and pFlag-N17-Rac1B (D,F); pFlag-p95-C3 and pFlag-V12-Rac1B (G-I,N,O); pFlag-p95-N4 and pFlag-V12-Rac1B (J,K); pFlag-p95-C3, pCMV6m/Pak1, and pFlag-V12-Rac1B (R,S); pFlag-p95-C3, pXJ40-HA-ßPIX, and pFlag-V12-Rac1B (T,U). Cells were fixed and processed for indirect immunofluorescence one day after transfection. The same field is shown in (C,E), (D,F), (G,H), (J,K), (L,M), (N,O), (P,Q), (R,S), and (T,U). Bars, 10 µm. V12-Rac induces the localization of both p95-C2 (B,C,E) and p95-C3 (G,H) at the cell periphery, together with paxillin (L-O), Pak (R, S), and Pix (T,U). V12-Rac does not influence the localization of p95-N4 (J,K).

View larger version (134K): [in a new window]   Fig. 10. Localization of p95-C2 at the cell periphery. COS7 cells transfected for 20 hours with pFlag-p95-C2 were incubated for 3 hours without serum, before incubation for 10 minutes with medium containing 50 ng/ml of PDGF (B-E). After fixation, cells were immunostained for p95-C2 (A,B,D), and incubated with fluorescently-labelled phalloidin (C,E). Same fields are shown in B,C, and in D,E. Bar, 10 µm.

View larger version (19K): [in a new window]   Fig. 11. Model for the role of the p95 complex in membrane recycling from the endocytic compartment. P95-APP1 interacts with PIX which in turn interacts with PAK. Moreover, the C-terminal portion of p95-APP1 can interact with paxillin, and induce paxillin relocalization away from focal adhesions. Both PIX and the ankyrin repeats of p95-APP1 may induce recruitment of the p95 complex to the endosomal compartment. In particular, PIX is required for the recruitment to Rab11-positive recycling endosomes (R.E.). Internalized membrane can normally be recycled back to the plasma membrane (PM) from the Rab11-positive recycling compartment. According to this model, the lack of a functional ARF-GAP domain may interfere with membrane recycling by preventing ARF-mediated vesicle budding from the recycling compartment, thus inducing an abnormal accumulation of large Rab11-positive vesicles. By contrast, activation of Rac at the cell surface may induce translocation of the p95 complex, and possibly of recycling vesicles, to the cell surface via the interaction with the Rac effector PAK. This would result in the transport to the cell periphery of new focal adhesion components (paxillin) and of part of the machinery required for Rac-mediated actin assembly (PAK, PIX). See text for more details.