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First published online 15 January 2008
doi: 10.1242/jcs.008037

Journal of Cell Science 121, 369-378 (2008)
Published by The Company of Biologists 2008
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Multiple regulatory inputs converge on cortactin to control invadopodia biogenesis and extracellular matrix degradation

Inmaculada Ayala1, Massimiliano Baldassarre1,*, Giada Giacchetti1, Giusi Caldieri1, Stefano Tetè2, Alberto Luini3 and Roberto Buccione1,§

1 Tumour Cell Invasion Laboratory, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, S. Maria Imbaro (Chieti), Italy
2 Department of Oral Sciences, University "G. D'Annunzio", 66013, Chieti, Italy
3 Membrane Traffic Laboratory, Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, S. Maria Imbaro (Chieti), Italy


Figure 1
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  		Fig. 1. Main features of invadopodia and effect of BB-94 treatment. (A) Triple staining of A375MM cells grown on crosslinked FITC-conjugated matrix for 16 hours and then fixed and stained with anti-cortactin (A) and Alexa Fluor 633-phalloidin (B), or anti-dynamin 2 (E) and anti-phosphotyrosine (F). All these proteins colocalized precisely to ECM degradation patches (C and G, arrows). (B) Anti-cortactin (A,E) and Alexa Fluor 633-phalloidin (B,F) labeling of cells grown for 16 hours on fluorescent matrix (C,G) in the continuous presence (+BB94) (A-D) and 3 hours after washout (–BB94) of the inhibitor (E-H). Scale bars: 10 µm.

 

Figure 2
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  		Fig. 2. Depletion of cortactin by RNA interference. (A) Lysates of A375MM cells 72 hours after mock or cortactin-specific siRNA transfection were subjected to SDS-PAGE, transferred to nitrocellulose and probed with anti-cortactin and anti-GAPDH as a loading control. (B) The relative amount of cortactin in mock-transfected and knockdown cells was measured. A 95% reduction in the amount of cortactin was observed. (C) Immunofluorescence labeling with anti-cortactin and phalloidin of cortactin siRNA-treated A375MM cells. Depletion of cortactin blocks the formation of invadopodia and hence, matrix degradation. (D) Transfection of cortactinWT 48 hours after knockdown. Normal invadopodia form and degrade the extracellular matrix. Scale bars: 10 µm.

 

Figure 3
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  		Fig. 3. Schematic diagram of the cortactin constructs used in this study. Cortactin features a N-terminal acidic domain (NTA) which specifically binds the Arp2/3 complex and is followed by a variable number of 37 amino acids repeats, the fourth of which binds F-actin. After an {alpha}-helix of undefined function, there is a proline-rich domain (PRD). Finally, the C-terminal is a Src-homology 3 domain (SH3) able to bind the proline-rich domain of several binding partners. The arrows indicate the positions of the mutated sites.

 

Figure 4
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  		Fig. 4. Analysis of the cortactin functional domains required for ECM degradation at invadopodia. (A) A375MM cells transfected with mutant forms of cortactin, display a significantly reduced ability to degrade the ECM compared with control cells transfected with cortactinWT. Data represent the mean ± s.d. of three independent experiments. Statistical significances were evaluated by Student's t-test: wild type vs {Delta}NTA, W22A and {Delta}SH3, P<0.006. (B) Transient re-expression of DsRed-tagged mutant and deleted forms of cortactin following knockdown as determined by immunoblotting. A typical experiment is shown. Line indicates endogenous cortactin. (C) Transient re-expression of FLAG-tagged mutant forms of cortactin following knockdown as determined by immunoblotting. A typical experiment is shown.

 

Figure 5
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  		Fig. 5. Tyrosine phosphorylation of cortactin is required for ECM degradation at invadopodia. (A) A375MM cells transfected with cortactinY421,466,482F display a significantly reduced ability to degrade the ECM compared with control cells transfected with cortactinWT. By contrast, cells transfected with the pseudophosphorylated cortactinY421D increased their capacity to degrade the matrix. Data represent the mean ± s.d. of three independent experiments. Statistical significance was evaluated by Student's t-test: wild type vs Y421D and Y421,466,482F, P<0.002. (B) Representative images of A375MM cells transfected with cortactinY421D 48 hours after cortactin depletion. Typically, cortactinY421D localizes to invadopodia. Scale bar: 10 µm.

 

Figure 6
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  		Fig. 6. Serine phosphorylation of cortactin is required for ECM degradation at invadopodia. (A) A375MM cells transfected with cortactinS405,418D display a remarkably increased ability to degrade the ECM compared with control cells transfected with cortactinWT. By contrast, transfection of cortactinS405,418A markedly reduced ECM degradation. Data represent the mean ± s.d. of three independent experiments. Statistical significance evaluated by Student's t-test: wild type vs S405,418D, P<0.01; wild type vs S405,418A, P<0.005. (B) Representative image of A375MM cells transfected with cortactinS405,418D 48 hours after cortactin depletion. Typically cortactinS405,418D localizes to invadopodia. (C) A375MM cells transfected with the autoinhibitory domain of PAK (AID) display a reduced ability to degrade the matrix compared with mock-transfected cells. Data represent the mean ± s.d. of three independent experiments. Statistical significance was evaluated by Student's t-test: control vs autoinhibitory domain of PAK P<0.0001. (D) A375MM cells were transfected with cortactinWT, nonphosphorylatable cortactinS113A and pseudophosphorylated cortactinS113D. The areas of degradation were then quantified. Data represent the mean ± s.d. of three independent experiments. Statistical significance was evaluated by Student's t-test: wild type vs S113A and S113D, P<0.004. (E) Representative immunofluorescence image of A375MM cells transfected with cortactinS113D 48 hours after cortactin knockdown labeled with anti-FLAG antibodies. CortactinS113D clearly localizes to ECM degradation patches at invadopodia. Scale bars: 10 µm.

 

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© The Company of Biologists Ltd 2008