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First published online 25 July 2006
doi: 10.1242/jcs.03076

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Palladin binds to Eps8 and enhances the formation of dorsal ruffles and podosomes in vascular smooth muscle cells

¹ Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, NC 27 27599, USA
² IFOM Istituto FIRC di Oncologia Molecolare Via Adamello 16, 20139, Milan, Italy
³ Department of Experimental Oncology, Istituto Europeo di Oncologia (IEO), Via Ripamonti 435, 20141, Milan, Italy
⁴ Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, NC 27 27599, USA

View larger version (101K): [in a new window]   Fig. 1. Palladin localizes to PDGF-induced membrane ruffles. A7r5 cells were plated on fibronectin overnight and serum-starved for 2 hours prior to growth factor treatment. The addition of PDGF led to the induction of dynamic, actin-based membrane protrusions (indicated by arrows). After fixation, endogenous palladin was detected in these structures by immunofluorescence. Co-labeling with TRITC-phalloidin and polyclonal anti-palladin antibody reveals that palladin co-localizes with filamentous actin in ruffles and along stress fibers. Top: low magnification image. Bottom: high magnification image to show detail. Bar, 10 µm.

View larger version (66K): [in a new window]   Fig. 2. Palladin knockdown decreases PDGF-induced ruffle formation. (A) A7r5 cells transfected with control pSuper RNAi and pSuper RNAi targeting palladin were plated on fibronectin overnight and serum-starved for 2 hours prior to PDGF treatment. Cells were fixed, permeabilized and stained with TRITC-phalloidin. Transfected cells (green fluorescence) were detected by the presence of GFP encoded in the pSuper vector. (B) The proportion of cells developing ruffles after PDGF stimulation is shown for cells transfected with palladin siRNA (knockdown, KD, 10±2%) and transfected with control siRNA (C, 50±5%). Results are representative of three independent experiments in which at least 100 transfected cells were counted. Bar, 10 µm.

View larger version (72K): [in a new window]   Fig. 3. Palladin localizes to PDBu-induced podosomes. A7r5 cells were plated on fibronectin and treated with the phorbol ester PDBu. After fixation, endogenous palladin was detected by immunofluorescence. Co-labeling with TRITC-phalloidin and polyclonal anti-palladin antibody reveals that palladin co-localizes with actin. Top: low magnification image. Bottom: high magnification image to show detail.

View larger version (75K): [in a new window]   Fig. 4. Palladin knockdown decreases PDBu-induced podosome formation. (A) A7r5 cells transfected with control pSuper RNAi and pSuper RNAi targeting palladin were plated on fibronectin overnight and treated with PDBu. Cells were fixed, permeabilized and stained with TRITC-phalloidin. Transfected cells (green fluorescence) were detected by the presence of GFP encoded in pSuper. Top: low magnification image. Bottom: high magnification image to show detail. (B) The proportion of cells developing podosomes after PDBu stimulation is shown for cells transfected with palladin siRNA (knockdown, KD, 20±6%) and transfected with control siRNA (C, 42±8%). Results are representative of three independent experiments in which at least 100 transfected cells were counted.

View larger version (43K): [in a new window]   Fig. 5. Palladin specifically interacts with Eps8. (A) Schematic representation of the palladin protein used as bait. The amino acid positions are indicated by the flanking numbers. The structural domains are shown as boxes: the N-terminal half of the molecule contains a serine-rich (S) and a proline-rich (P) domain, and the C-terminal half of the molecule contains three tandem repeats of a domain called `IgC2'. (B) Schematic representation of full-length Eps8 and the truncated form found in the yeast two-hybrid screen. The amino acid positions are indicated as in A. The structural domains are shown as boxes: a phosphotyrosine-binding domain (PTB), an SH3 domain and an effector region (ER). (C) Palladin and Eps8 form complexes in cultures cells. Left panel: alladin was immunoprecipitated with 1E6 monoclonal antibody from lysed Swiss 3T3 fibroblasts, and the samples were probed with anti-Eps8 monoclonal antibodies to determine whether Eps8 co-precipitates with palladin. Whole cell lysate was run as positive control, and a sample that contained no primary antibody (beads alone) was run as negative control. Right panel: Eps8 was immunoprecipitated with anti-Eps8 antibody and blotted for palladin, also with whole cell lysate and beads alone as positive and negative controls, respectively. Note that Eps8 was detected in the palladin immunoprecipitate and palladin was detected in the Eps8 immunoprecipitate. L, whole cell lysate; C, beads alone; Ab, antibody used for IP. (D) Western blot analysis from cytosolic extracts of A7r5 cells treated with PDGF. Cells were untreated (SS, serum starvation) or treated with PDGF for 5, 10 and 30 minutes (5', 10' and 30'). Blots were probed with anti-palladin 1E6 monoclonal antibody. (E) Co-immunoprecipitation of palladin and Eps8 from cytosolic extracts of Eps8 null mouse embryonic fibroblasts (MEF Eps8-/-) and rescued MEF (MEF Eps8-/- + myc-Eps8). Cells were untreated (SS) or treated with PDGF for 10 and 30 minutes (10' and 30'). Eps8 antibody was used for immunoprecipitation. Blots were probed with anti-Eps8 Abs or anti-palladin 1E6 monoclonal antibody.

View larger version (25K): [in a new window]   Fig. 6. Yeast two-hybrid analysis of palladin-Eps8 interaction. (A) Mapping of the palladin-binding site in Eps8. Yeast cells were co-transformed with various Eps8 fragments in prey vector and full-length palladin in bait vector. (B) Mapping of the Eps8 binding site in palladin. Palladin C-terminal, N-terminal and overlapping N-terminal constructs were tested for interaction with Eps8. Growth was estimated after 5 to 7 days of incubation at 30°C. The formation of colonies secreting X-alpha-gal was taken as an indication of an interaction between the expressed proteins and is noted by `+'. No interaction between expressed proteins is noted with `-'. Boxes shown are the regions mapped to be essential for the interaction in each analysis. Controls were systematically performed for each construct with the opposite vectors without insert. Each experiment was repeated at least twice.

View larger version (107K): [in a new window]   Fig. 7. Palladin co-localizes with Eps8. A7r5 cells were plated on fibronectin-coated coverslips and were either incubated for 2 hours in serum-free media prior to treatment with PDGF (A) or treated with the phorbol ester PDBu (B). Cells were fixed and immunolabeled with polyclonal anti-palladin antibody and monoclonal anti-Eps8 antibody. The two images were merged (overlay) to show the relative localization of palladin (green) and Eps8 (red). Top: low magnification image. Bottom: high magnification image to show detail.

View larger version (15K): [in a new window]   Fig. 8. Rac activity is decreased in palladin knockdown cells. (A) Western blot analysis of whole cell lysates of HeLa cells transfected with control siRNA (C) or siRNA targeting palladin (knockdown, KD). Active Rac was specifically pulled down from cell lysates containing equal amounts of proteins with immobilized recombinant PAK-CRIB and analyzed by western blotting using anti-Rac antibody. A representative experiment is shown. (B) The level of active Rac was quantified by densitometric analysis of western blots and the amount of active Rac was normalized to the amount of total Rac. Rac activation was quantified from three independent assays.