SWAP-70 identifies a transitional subset of actin filaments in motile cells

Abstract

Functionally different subsets of actin filament arrays contribute to cellular organization and motility. We report the identification of a novel subset of loose actin filament arrays through regulated association with the widely expressed protein SWAP-70. These loose actin filament arrays were commonly located behind protruding lamellipodia and membrane ruffles. Visualization of these loose actin filament arrays was dependent on lamellipodial protrusion and the binding of the SWAP-70 PH-domain to a 3'-phosphoinositide. SWAP-70 with a functional pleckstrin homology-domain lacking the C-terminal 60 residues was targeted to the area of the loose actin filament arrays, but it did not associate with actin filaments. The C-terminal 60 residues were sufficient for actin filament association, but they provided no specificity for the subset of loose actin filament arrays. These results identify SWAP-70 as a phosphoinositide 3-kinase signaling-dependent marker for a distinct, hitherto unrecognized, array of actin filaments. Overexpression of SWAP-70 altered the actin organization and lamellipodial morphology. These alterations were dependent on a proper subcellular targeting of SWAP-70. We propose that SWAP-70 regulates the actin cytoskeleton as an effector or adaptor protein in response to agonist stimulated phosphatidylinositol (3,4)-bisphosphate production and cell protrusion.

Figure 5.

SWAP-70 localizes to loose actin filament arrays located behind actively extending lamellipodia. B16F1 cells were transfected with GFP-SWAP-70, replated on laminin-coated coverslips and images were acquired 1 h (A) or 4 h (B and C) after replating. Corresponding fluorescence and phase contrast images are shown. (A) Spreading cell. The lamellipodium protrudes, achieves its maximal size at the 600-s time point and then collapses. (B) Slowly migrating cell, which is changing the direction of migration. (C) Ruffling cell with SWAP-70 localizing to the cell edge of retracting area. Bars, 10 μm. See also Videos 1, 2, and 3.

Figure 1.

SWAP-70 is widely expressed. Equal amounts of protein from different adult rat tissues and human and mouse cell homogenates were immunoblotted with the affinity-purified SWAP-70 antibody GK2. Tissues and cells are indicated as described above each lane. Molecular mass standards in kilodaltons are shown on the left.

Figure 2.

Endogenous SWAP-70 associates with a subset of loose actin filament arrays. B16F1 cells were plated on 25 μg/ml laminin for 5 h before paraformaldehyde (A and B) or glutaraldehyde (C and D) fixation and double labeling with SWAP-70 antibody GK2 (A and C) and phalloidin (B and D). Areas boxed in C and D are shown at higher magnification in E and F, respectively. Arrows in E and F show the colocalization of SWAP-70 (E) with fine actin filament bundles (F). Bars, A–D, 10 μm; E and F, 1.5 μm.

Figure 3.

SWAP-70 does not localize on myosin II-containing actin fibers. B16F1 cells were transfected with GFP-SWAP-70 as described in MATERIALS AND METHODS and fixed for 3 min in 4% PFA, 0.1% Triton X-100 followed by fixation in 4% PFA for 30 min. Cells were labeled for myosin II by indirect immunofluorescence by using a secondary antibody coupled to Alexa 568. GFP-SWAP-70 (A), myosin II (B), and merge of the two stainings (C). Bar, 10 μm.

Figure 4.

GFP-constructs. (A) Thirty hours after transfection with GFP-SWAP-70, B16F1 cells were replated on laminin-coated coverslips and after an additional 5 h fixed with paraformaldehyde and stained with Alexa 594-phalloidin. Bars, 10 μm. (B and C) B16F1 cells were transfected with different GFP-SWAP-70-constructs for 30 h and then analyzed by Western blotting with polyclonal affinitypurified SWAP-70 antibody GK2 (B) or monoclonal GFP antibody (C). Equal amounts of protein (B, 10 μg; C, 40 μg) from cell homogenates were loaded. Cells were transfected with the following constructs: lanes 1 and 3: pEGFP-C1; lanes 2 and 4: GFP-SWAP-70; lane 5: GFP-SWAP-R230C; lane 6: GFP-SWAP-RR223,224EE; lane 7: GFP-SWAP(205-585); lane 8: GFP-SWAP(1-313) and lane 9: PH(205-313)GFP. The *a–f indicates the size of the different constructs: a (∼100 kDa): GFP-SWAP-70; b (70 kDa): endogenous SWAP-70; c (∼75 kDa): GFP-SWAP(205-585); d (∼67 kDa): GFP-SWAP(1-313); e (∼43 kDa): PH(205-313)-GFP; and f (∼30 kDa): GFP. (D) Schematic representation of SWAP-70 GFP-fusion proteins. (E) Residues conserved in PH domains binding to PI(3,4)P₂ and PI(3,4,5)P₃ (Isakoff et al., 1998) are aligned to the SWAP-70 sequence. Mutated residues in constructs R230C and RR223,224EE are indicated in bold.

Figure 6.

The PH domain of SWAP-70 binds phosphoinositides in vitro. GST-SWAP-70 constructs were expressed in E. coli and purified. The PIP-strips were incubated with different fusion proteins indicated on top of each PIP-strip at concentrations of 0.5 μg/ml (SWAP-wt, SWAP-R/C, SWAP-RR/EE, and PH-RR/EE) or 0.01 μg/ml (PH-wt and PH-R/C). Bound fusion proteins were detected by indirect immunostaining. The GST-SWAP-70 fusion proteins were detected by GK2 antibody and the GST-PH fusion proteins with GST antibody. The phospholipids (100 pmol) spotted on PIP-strips were as follows: phosphatidylinositol (PtdIns), phosphatidylcholine (PC), inositol(1,3,4,5)-tetraphosphate [I(1,3,4,5)P4], phosphatidylinositol(3,5)-bisphosphate [PI(3,5)P2], phosphatidylinositol (3)-phosphate [PI(3)P], phosphatidic acid (PA), phosphatidylinositol (4)-phosphate [PI(4)P], phosphatidylinositol(4,5)-bisphosphate [PI(4,5)P2], phosphatidylinositol (5)-phosphate [PI(5)P], phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol(3,4,5)-trisphosphate [PI(3,4,5)P3], and phosphatidylinositol(3,4)-bisphosphate [PI(3,4)P2].

Figure 7.

The PH-domain is necessary for proper localization of SWAP-70 to loose actin filament arrays, but it is not sufficient. B16F1 cells were transfected with GFP-SWAP-RR/EE (A and B) and PHGFP (E and F) or cotransfected with CFP-SWAP-70 (C) and YFP-SWAP-70R230C (D). GFP-SWAP-70-RR/EE– and PH-GFP–transfected cells were stained with Alexa 594-phalloidin (B and F). The cell shown in C and D was treated with AlF₄^– for 20 min. before fixation. Fluorescence images of representative cells are shown. In A–D, epifluorescence images are shown and in E, F a 0.4-μm confocal section. Bars, 10 μm.

Figure 8.

Truncation of both, the N-terminal region or the C-terminal region of SWAP-70 abolishes the localization to the subset of loose actin filament arrays. The C-terminal 60 residues contain F-actin–targeting information. B16F1 cells were transfected with GFP-SWAP(1-313) (A and B), GFP-SWAP(205-585) (C and D), or GFP-SWAP(526-585) (G and H) and double stained with Alexa 594-phalloidin (B, D, and H). The cell in C and D was fixed in the presence of Triton X-100 to improve visibility of colocalization of GFP-SWAP(205-585) with F-actin. The arrows in C and D point at the regions enlarged in the insets. In the insets the lack of GFP-SWAP(205-585) in focal contacts is shown. (E and F) B16F1 cells were cotransfected with CFP-SWAP-70 (E) and YFP-SWAP(1-525) missing the C-terminal 60 amino acids (F). Cells were fixed and viewed in the fluorescence microscope. Bars, 10 μm.

Figure 9.

Overexpression of SWAP-70 alters the actin organization. GFP-SWAP-70 (A and D) was overexpressed in HtTa-1 HeLa cells (A–C) and B16F1 cells (D–F). Cells were fixed, permeabilized, and double stained with Alexa 594-phalloidin (B and E). Corresponding phase contrast images are shown in C and F. Bars, 10 μm. Note that GFP-SWAP-70–overexpressing cells show either a loss (HtTa-1 HeLa) of lamellipodia or an altered lamellipodial morphology (B16F1).