Formin homology domains of Daam1 bind to Fascin and collaboratively promote pseudopodia formation and cell migration in breast cancer

Figures

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  FIGURE 1. Knockdown of Fascin inhibits the assembly of actin filaments and the formation of pseudopodia. A and B, MCF‐7 and MDA‐MD‐231 cells were transfected with negative control (NC) and siRNA targeting Fascin (si‐Fascin). The expression of Fascin was determined by Western blotting. C and D, The morphology of actin filaments showed a short and curved state in Fascin‐knockdown MCF‐7 cells compared with NC cells. The number of actin filaments (length > 10 μm) and the longest actin filament in Fascin‐downregulated MCF‐7 cells were counted. Actin filaments were labelled with red fluorescently labelled phalloidin. Endogenous Fascin was incubated with anti‐Fascin antibody and green fluorescent protein secondary antibody. The nucleus was stained with DAPI (blue). Bar, 10 μm. E and F, MDA‐MD‐231 cells transfected with si‐Fascin and/or 3HA‐Fascin were detected for invadopodia formation in a 3.0 μm porous Boyden chamber. Invadopodia on the lower side of the membrane were treated with cortactin antibody and counted per field of microscope. Bar, 10 μm. Objective lens, magnification, ×40; numerical aperture, 0.95. *P < .05. **P < .01. ****P < .0001

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  FIGURE 2. The expression of Fascin is upregulated in BrCa tissues, and downregulated Fascin restrains cell migration. A and B, In contrast to Fascin expression in paracarcinoma tissues, Fascin was highly expressed in BrCa tissues. Fascin was visualized with strongly positive staining in the cytoplasm. Bar, 500 pixel. Objective lens, magnification, ×20. The boxes were shown the local enlargement tissues. The immunoreactivity score (IRS) was calculated by multiplying the percentage of positive cells by the staining intensity. C and D, MDA‐MD‐231 cells transfected with siRNA targeting Fascin (si‐Fascin) and/or 3HA‐Fascin were allowed to migrate for 12 h. The wound healing area was measured and counted. Bar, 10 μm. Objective lens, magnification, ×20. E and F, MDA‐MD‐231 cells transfected with si‐Fascin and/or 3HA‐Fascin migrated for 6 h in Boyden chambers. The number of migratory cells per field was microscopically counted. Bar, 10 µm. Objective lens, magnification, ×20. *P < .05. **P < .01. ****P < .0001

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  FIGURE 3. Daam1 interacts with Fascin and is significantly coexpressed in BrCa tissues. A, Schematic presentation of the domain structures of full‐length human Daam1 (Daam1‐full), GBD‐FH1, FH2 and FH2‐DAD. B, Daam1‐full, GBD‐FH1, FH2, FH2‐DAD and human Fascin were inserted into pCDNA 3.1‐3FLAG or pCDNA 3.1‐3HA vectors. The expression of exogenous proteins in BrCa cells transfected with the indicated constructs was examined by Western blotting. C and D, Endogenous Daam1 and Fascin coimmunoprecipitated with each other with the anti‐Daam1 or anti‐Fascin antibody. The whole‐cell lysates were analysed by immunoblotting with the indicated antibodies. **P < .01. E and F, For in vitro binding assay, MCF‐7 cells expressing endogenous Daam1 or transfected with 3FLAG‐Daam1‐full were lysed and then mixed with bacterially expressed His‐Fascin. The pull‐down products and all inputs were immunoblotted with the indicated antibodies. G and H, Daam1 and Fascin showed similarly strong positive staining in BrCa immunohistochemical sections. Bar, 10 μm. Objective lens, magnification, ×20. The coexpression relationship was analysed by correlation analysis. r = .5268, P < .01

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  FIGURE 4. Daam1 binds to Fascin via FH domains. A, The lysates of MCF‐7 cells transiently expressing 3FLAG‐Daam1‐full, 3FLAG‐GBD‐FH1, 3FLAG‐FH2 and 3FLAG‐FH2‐DAD were mixed with bacterially expressed His‐Fascin and examined by immunoblotting with anti‐FLAG antibody. B‐D, The morphology of actin filaments in MCF‐7 cells expressed with the indicated constructs was visualized by immunofluorescence microscopy. The number of actin filaments (length > 10 μm) per cell and the length of the longest actin filaments were counted. Bar, 5 μm. E and F, MDA‐MD‐231 cells transfected with the indicated constructs migrated for 6 h in Boyden chambers. The number of migratory cells per field was counted. Bar, 10 μm. Objective lens, magnification, ×20. G and H, MDA‐MD‐231 cells were transiently transfected with the indicated constructs. Pseudopodia (invadopodia) were allowed to extend in 3.0 μm porous Boyden chambers. Invadopodia on the lower side of the membrane were incubated with cortactin antibody and counted per field of microscope. Bar, 10 μm. Objective lens, magnification, ×40; numerical aperture, 0.95. *P < .05. ***P < .001. ****P < .0001

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  FIGURE 5. Daam1 and Fascin recruit each other, and they cooperatively mediate actin filament assembly and pseudopodia formation. A‐D, Endogenous Daam1 (green) and Fascin (blue) in MCF‐7 cells were colocalized with actin filaments and pseudopodia (filopodia, red). Bar, 5 μm. The boxed region shows the enlargement of local actin filaments, and white arrowheads indicate colocalization. Bar, 100 pixel. Fluorescence intensity profiles of Daam1, Fascin and actin were measured at the positions marked by arrows in merged insets. E‐G, Fascin‐silenced MCF‐7 cells (Fascin‐siRNA) were immunostained with antibodies against Daam1 (green) and Fascin (blue) and compared to the rescue group (Fascin‐siRNA + 3HA‐Fascin). The actin filaments were stained with phalloidin (red). Bar, 5 μm. The boxed region showed the local enlargement state. Bar, 5 pixel. H‐J, Daam1‐silenced MCF‐7 cells (Daam1‐shRNA) were immunostained with antibodies against Daam1 (cyan) and Fascin (blue) compared to the rescue group (Daam1‐shRNA + 3FLAG‐Daam1‐full). ShRNA targeting Daam1 was labelled with GFP (green), and actin filaments were labelled with phalloidin (red). Bar, 5 μm. The boxed region showed the local enlargement state. Bar,…

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  FIGURE 6. Schematic diagram of the mechanism by which Daam1 and Fascin collaborate to promote cell motility in BrCa cells