WISp39 binds phosphorylated Coronin 1B to regulate Arp2/3 localization and Cofilin-dependent motility.
WISp39 knockdown (KD) resulted in the loss of directional motility of mammalian cells and profound changes in cell morphology, including the loss of a single leading edge.WISp39 KD-induced morphological changes could be rescued by overexpression of Coronin 1B together with a constitutively active Cofilin mutant.We conclude that WISp39 associates with Hsp90, Coronin 1B, and SSH to regulate Cofilin activation and Arp2/3 complex localization at the leading edge.
Affiliation: Sanford-Burnham Medical Research Institute, La Jolla, CA 92037.
- Actin Depolymerizing Factors/genetics/metabolism*
- Actin-Related Protein 2-3 Complex/metabolism*
- Microfilament Proteins/biosynthesis/metabolism*
- Cell Line, Tumor
- Cell Movement/genetics
- Enzyme Activation/genetics
- HEK293 Cells
- HSP90 Heat-Shock Proteins/metabolism
- HeLa Cells
- Phosphoprotein Phosphatases
- Protein Binding
- RNA Interference
- RNA, Small Interfering
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fig6: WISp39 KD decreases the localization of Arp2/3 complex and increases the generation of free barbed ends at the cell leading edge. (A) WISp39 KD decreases Arp2/3 complex localization at the cell leading edge. Immunolocalization of the Arp2/3 subunit p34-Arc and F-actin phalloidin staining in cells transfected for 48 h with control or WISp39 siRNA. U2OS cells were transfected with WISp39 siRNA in combination with a GFP plasmid expressing histone H2B to visualize transfected cells. After 48 h, the cells were fixed, and immunofluorescence images were acquired as described in Materials and methods. Insets in the first image show a cell expressing GFP-H2B, cotransfected with the siRNA. Magnified regions are shown in the right-most image. (B) Quantification of fluorescence intensity (±SEM) of F-actin, p34-Arc, and ratio of p34-Arc/F-actin in control and WISp39-depleted cells, measured from the cell edge (0) to the cell center (7 µm). Bands of constant distance to the cell edge were constructed, and individual fluorescence intensities were accumulated and averaged in each band to produce graphs of fluorescence intensities versus distance to the cell edge (Fig. S5). The data shown represent one experiment and are averaged from n ≥ 11 cells for each condition. Only cells with a spread morphology have been included. Student’s t test; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 compared with control siRNA cells. The experiment was repeated three times, with similar results. (C) G-actin incorporation marking free barbed ends and F-actin phalloidin staining in cells transfected for 48 h with control or WISp39 siRNA. U2OS cells were transfected with WISp39 siRNA in combination with GFP plasmid expressing histone H2B for 48 h, and immunofluorescence localization of barbed ends was performed by live-cell microscopy. Insets in the first image show the expression of H2B, cotransfected with the siRNA. Magnified regions are shown in the right-most image. WISp39 depletion induces the formation of free barbed filament ends. (D) Quantification of fluorescence intensity of F-actin, free barbed ends, and the ratio of free barbed ends: F-actin in control and WISp39 KD cells, measured from the cell edge (distance = 0) to the cell center (7 µm). Quantitation was performed as in B. The data shown represent one experiment and are averaged from n ≥ 15 cells for each condition (±SEM). Only cells with a spread morphology have been included. Student’s t test; *, P ≤ 0.05; ***, P ≤ 0.001 compared with control siRNA cells. The experiment was repeated four times, with similar results. The mean fluorescent F-actin intensity is elevated slightly relative to B, and this variation may arise because the analysis in B was performed on fixed cells. a.u., arbitrary unit. Bars: (left images and insets) 15 µm; (zoom images) 5 µm.
The phosphorylation state of Coronin 1B determines its binding to the Arp2/3 complex (Fig. 5 A; Cai et al., 2005), and WISp39 influences the phosphorylation of Coronin 1B. We therefore investigated the effect of WISp39 on Arp2/3 complex distribution at the leading edge. WISp39 colocalizes with the p34-Arc subunit of the Arp2/3 complex at the leading edge, as indicated by the robust correlation coefficients obtained in linear regression analysis (Fig. S3). We next determined whether the loss of WISp39 would have an effect on Arp2/3 distribution at the leading edge. We consistently found less p34-Arc subunit at the leading edge of WISp39 KD cells compared with controls, irrespective of the cell morphology (Fig. 6, A and B; and Fig. S4). This result was unexpected, as increased phosphorylation of Coronin 1B in WISp39 KD cells should reduce the ability of Coronin 1B to bind and inhibit the Arp2/3 complex, resulting in increased localization of active Arp2/3 complex to the leading edge. As WISp39 affects Arp2/3 complex localization at the leading edge, we next checked whether the barbed ends were affected by the loss of WISp39. Despite the decreased Arp2/3 complex localization, WISp39 depletion increased the concentration of barbed ends relative to total actin at the leading edge (Fig. 6, C and D). Our data are consistent with a previous study showing increased free barbed ends in Coronin 1B KD cells (Cai et al., 2007).