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First published online 17 January 2006
doi: 10.1242/jcs.02767

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Direct binding of SWAP-70 to non-muscle actin is required for membrane ruffling

Sayoko Ihara, Tsutomu Oka^* and Yasuhisa Fukui

Division of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

View larger version (35K): [in a new window]   Fig. 1. The C-terminal region of SWAP-70 binds to non-muscle actin in an isospecific manner. (A) The structure of SWAP-70 and its truncation mutants. The amino acid sequence of the putative actin-binding region in SWAP-70 was aligned with those of the homologous region seen in proteins of the gelsolin family. Basic amino acids are shown with a gray background; PH, pleckstrin-homology domain. (B) Binding of the C-terminal region of SWAP-70 to F-actin. His–SWAP-70(448-585) or His–SWAP-70(448-564) was mixed with or without (–) 10 µM polymerized actin derived from human platelets (non-muscle) or rabbit skeletal muscle (muscle), and ultracentrifuged. Proteins in the supernatant (S) and the pellet (P) were analyzed by SDS-PAGE followed by staining with Coomassie Blue. (C) Quantitative analysis for binding of the C-terminal region of SWAP-70 to F-actin. A co-sedimentation assay was performed by mixing of 5 µM polymerized non-muscle actin with various amounts of His–SWAP-70(448-585) at the indicated final concentrations. (D) Similar experiments as in C with a wide range of concentrations of His–SWAP-70(448-585) were performed, and the amounts of protein on the gel were quantified by an imaging analyzer.

View larger version (28K): [in a new window]   Fig. 2. The actin-binding property of SWAP-70. (A) Binding of full-length SWAP-70 to F-actin. His–SWAP-70 or His–SWAP-70(1-564), lacking the actin-binding domain, was mixed with (+) or without (–) 10 µM polymerized non-muscle actin. Proteins in the supernatant (S) and the pellet (P) were analyzed by SDS-PAGE followed by Coomassie Blue staining. The band seen at the position of actin in the lane of actin (–) is the degradation product of His–SWAP-70. The same protein may be present in the lane of actin (+). (B) Quantitative analysis for binding of His–SWAP-70 to F-actin. A co-sedimentation assay was performed by mixing of 5 µM polymerized non-muscle actin with various amounts of His–SWAP-70 (0 µM to 5 µM). (C) Binding property of full-length SWAP-70. A co-sedimentation assay was performed by mixing of His–SWAP-70 with polymerized 10 µM actin derived from human platelets (non-muscle) or rabbit skeletal muscle (muscle). (D) Binding of the PH domain of SWAP-70 to F-actin. A co-sedimentation assay was performed by mixing of His–SWAP-70 PH with polymerized 10 µM muscle actin, non-muscle actin, or without (–) actin. (E) Quantitative analysis for binding of the PH domain of SWAP-70 to F-actin. A co-sedimentation assay was performed by mixing 5 µM polymerized non-muscle actin with various amounts of His–SWAP-70 PH (0 µM to 5 µM). Quantitative analysis was done as in B. (F) Actin-binding properties of the various mutants of SWAP-70. The structure of SWAP-70 and its truncation mutants is shown. All constructs were tagged with His at the N-terminus. The actin-binding property of each protein is summarized on the right.

View larger version (70K): [in a new window]   Fig. 3. The C-terminal portion of SWAP-70 colocalizes with F-actin. (A) The structure of SWAP-70 and its truncation mutants; all constructs were tagged with GFP at the N-terminus. PH, pleckstrin-homology domain. (B) COS7 cells expressing GFP–SWAP-70(448-585), GFP–SWAP-70(448-564), or GFP–SWAP-70 PH were fixed and stained with Phalloidin to visualize F-actin (red), followed by analysis with confocal microscopy. The merged image of SWAP-70 and F-actin is also shown (`merge'). Areas boxed in the panels of GFP–SWAP-70(448-585) are shown at higher magnification in left upper panels. Bar, 20 µm. (C) COS7 cells expressing GFP–SWAP-70 were treated with or without EGF for 5 minutes and fixed with Phalloidin to visualize F-actin (red), followed by analysis with a confocal microscopy. The x/z planes shown in the upper panel are the vertical sections along the white lines shown in the middle part of the panels. Stacked images of each planes of focus (marked as `stacked') and dorsal planes of focus (marked as `dorsal') are shown. Membrane ruffles can be observed clearly in the x/z plane or the dorsal plane (arrowheads). Bar, 20 µm.

View larger version (25K): [in a new window]   Fig. 4. The actin-binding ability of SWAP-70 through the C-terminal region is crucial for membrane ruffling induced by EGF. (A) COS7 cells expressing GFP or GFP–SWAP-70(1-564) were treated with (+) or without (–) EGF (100 ng/ml) for 5 minutes and stained with Phalloidin to detect F-actin. Localization of SWAP-70 (green) and F-actin (red) was observed under the confocal microscope. Stacked images are indicated by `S', and dorsal planes of focus by `D'. Membrane ruffles are indicated by arrowheads. Bar, 20 µm. (B) COS7 cells expressing GFP or GFP–SWAP-70(1-564) were treated with EGF (100 ng/ml) for 5 minutes and stained with Phalloidin to detect F-actin. Ruffling formation was quantified by scores defined in the Materials and Methods.

View larger version (45K): [in a new window]   Fig. 5. The actin-binding ability of SWAP-70 through the C-terminal region is crucial for membrane ruffling induced by dominant-active Rac. (A) COS7 cells were transfected with RFP-RacV12 together with GFP or GFP–SWAP-70(1-564). Localization of SWAP-70 (green) and Rac (red) was observed under the confocal microscope. Stacked images are indicated by `S', and dorsal planes of focus by `D'. Membrane ruffles (indicated by arrowheads) were observed in the cells that did not express GFP–SWAP-70(1-564), but not in those that expressed the protein. Bar, 20 µm. (B) Ruffling formation observed in the experiments illustrated in A was quantified by scores defined in the Materials and Methods (left panel). Formation of filopodia was monitored by similar analysis as in A by use of RFP-Cdc42V12 instead of RFP-RacV12 and quantified by scores defined in the Materials and Methods (right panel).

View larger version (58K): [in a new window]   Fig. 6. SWAP-70 binds to activated Rac. (A) Binding activity of SWAP-70 to Rho GTPases. Cell lysates from HEK-293T cells expressing GFP–SWAP-70 were incubated with GST-Rac loaded with GDP or GTPS, or GST-Rac free of guanine nucleotides (–), immobilized on glutathione-Sepharose beads. SWAP-70 trapped on the beads was analyzed by western blotting with anti-GFP antibody (upper panel). The filter was stained with Coomassie Blue to ensure that approximately equal amounts of GST proteins were present in each assay (bottom panel). A similar analysis was done using Cdc42 or Rho as probes. (B) Binding of SWAP-70 to Rac with mutations in the effector domain. Mutations shown in the figure were introduced into the effector domain of RacV12, a dominant-active form of Rac. The proteins were expressed as GST fusion proteins, and their binding activities to SWAP-70 were examined as described in A (upper panel). The amounts of GST fusion proteins used in the assay are shown in the bottom panel. Control, RacV12 without additional mutations.