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. 2013;8(3):e60528.
doi: 10.1371/journal.pone.0060528. Epub 2013 Mar 26.

IRSp53 mediates podosome formation via VASP in NIH-Src cells

Tsukasa Oikawa  1 , Hitomi Okamura, Franziska Dietrich, Yosuke Senju, Tadaomi Takenawa, Shiro Suetsugu
Affiliations

IRSp53 mediates podosome formation via VASP in NIH-Src cells

Tsukasa Oikawa et al. PLoS One. 2013.

Abstract

Podosomes are cellular "feet," characterized by F-actin-rich membrane protrusions, which drive cell migration and invasion into the extracellular matrix. Small GTPases that regulate the actin cytoskeleton, such as Cdc42 and Rac are central regulators of podosome formation. The adaptor protein IRSp53 contains an I-BAR domain that deforms membranes into protrusions and binds to Rac, a CRIB motif that interacts with Cdc42, an SH3 domain that binds to many actin cytoskeletal regulators with proline-rich peptides including VASP, and the C-terminal variable region by splicing. However, the role of IRSp53 and VASP in podosome formation had been unclear. Here we found that the knockdown of IRSp53 by RNAi attenuates podosome formation and migration in Src-transformed NIH3T3 (NIH-Src) cells. Importantly, the differences in the IRSp53 C-terminal splicing isoforms did not affect podosome formation. Overexpression of IRSp53 deletion mutants suggested the importance of linking small GTPases to SH3 binding partners. Interestingly, VASP physically interacted with IRSp53 in NIH-Src cells and was essential for podosome formation. These data highlight the role of IRSp53 as a linker of small GTPases to VASP for podosome formation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Essential role of IRSp53 in podosome formation, migration and invasion.
(A) Localization and involvement of IRSp53 in podosomes. NIH 3T3 cells transformed with constitutively active Src (NIH-Src cells) were transfected with the IRSp53 siRNA vector, and then stained with an anti-IRSp53 antibody (green). Actin filaments were visualized with phalloidin (red). The nuclear staining of IRSp53 represents non-specific signals, because the nuclear localization of GFP-IRSp53 was not obvious (Figure 2). Arrowheads indicate the cells with reduced IRSp53 expression, bearing no or tiny podosomes. Scale bar, 20 µm. (B) NIH-Src cells were transfected with control (Ctr) or IRSp53 siRNA and then cultured for 72 h. The cells were then subjected to immunoblot analyses with the indicated antibodies. (C) NIH-Src cells were transfected with control or IRSp53 siRNA and then cultured for 48 h. The cells were then stained with rhodamine–phalloidin to detect actin filaments, and the percentages of the cells with podosomes were counted. Data are means±SD from three independent experiments. Approximately 100 cells were counted in each determination. *P<0.05 (Student's t test). (D and E) The cell migration (D) or invasion (E) assay was performed as described in the materials and methods section, using serum as the chemo-attractant. Data are means±SD from three independent experiments. *P<0.05 (Student's t test).
Figure 2
Figure 2. IRSp53 splicing does not affect podosome formation.
(A) Restoration of podosome formation by the expression of each IRSp53 splicing isoform. NIH-Src cells were transfected sequentially with the IRSp53 siRNA vector and the expression vectors, and were stained with rhodamine-phalloidin (red) to visualize actin filaments and with anti-IRSp53 (blue). Scale bar, 20 µm. (B) Percentage of cells with podosomes in (A). Data are means±SD from three independent experiments. Approximately 100 cells were counted in each determination. **P<0.01 (Student's t test). The C-terminal amino acid residues of the IRSp53 splicing variants are also indicated.
Figure 3
Figure 3. Identification of the domains of IRSp53 required for podosome formation.
(A) The podosomes of cells expressing IRSp53 fragments. NIH-Src cells transfected with myc- or GFP-tagged expression vectors were observed by staining with anti-myc (green) or direct fluorescence of GFP, respectively. The cells were also stained with rhodamine-phalloidin (red) to visualize actin filaments. Scale bar, 20 µm. (B) Percentages of cells with podosomes expressing various IRSp53 mutants. The effects of the dominant-negative forms of Rac1 (Rac1 T17N) and Cdc42 (Cdc42 T17N) are also included. Data are means±SD from three independent experiments. Approximately 100 cells were counted in each determination. *P<0.05 versus myc-IRSp53 expressing cells (Student's t test). The domain structures of the construct used in this study are indicated in Fig. 5E.
Figure 4
Figure 4. Dispensable role of I-BAR domain for podosome formation.
(A) Restoration of podosome formation by the expression of the wild-type (WT) or BPM mutant of IRSp53. NIH-Src cells were transfected sequentially with IRSp53 siRNA and the expression vectors, and were stained with rhodamine-phalloidin (red) to visualize actin filaments and with anti-IRSp53 (blue). Scale bar, 20 µm. (B) Percentage of cells with podosomes in (A). Data are means±SD from three independent experiments. More than 100 cells were counted in each determination. *P<0.05 (Student's t test). (C) NIH-Src cells were transfected simultaneously with the FLAG-tagged constitutively active form of Rac (Rac1 G12V) and the GFP-tagged I-BAR domain of IRSp53. The R11E/Q23E mutant of I-BAR, which is defective in Rac binding, was used as a negative control for Rac binding . Anti-FLAG immunoprecipitates were then subjected to immunoblot analyses with the indicated antibodies.
Figure 5
Figure 5. Essential role of VASP in podosome formation.
(A) Interaction between IRSp53 and VASP in NIH-Src cells. Lysates of NIH-Src cells were subjected to immunoprecipitation with an antibody to VASP or control immunoglobulin G (IgG). The resulting precipitates, as well as the original cell lysates (Input), were then subjected to immunoblot analyses with the indicated antibodies. The arrowhead shows the band for IRSp53. (B) NIH-Src cells were transfected with control (Ctr) or VASP siRNA and then cultured for 72 h. The cells were then subjected to immunoblot analyses with the indicated antibodies. (C) NIH-Src cells were transfected with control or VASP siRNA, and actin filaments were visualized with phalloidin. Scale bar, 20 µm. (D) Data are means±SD from three independent experiments. More than 100 cells were counted in each determination. *P<0.05 (Student's t test). (E) Schematic diagram of the signaling to the actin cytoskeleton through IRSp53 in the podosome. The domain structures of the constructs used in this study are indicated.
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This work was supported by grants from the Funding Program for Next Generation World-Leading Researchers (NEXT Program) and the Astellas Foundation for Research on Metabolic Disorders to S.S. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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