Suppression of FAK Kinase Expression Decreases the Lifetime of Focal Adhesions and Inhibits Migration of Normal and Tumor Epitheliocytes in a Wound Healing Assay

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Focal adhesions (FAs) are mechanosensory structures that can convert physical stimuli into chemical signals guiding cell migration. There is a postulated correlation between FA features and cell motility parameters for individual migrating cells. However, which FA properties are essential for the movement of epithelial cells within a monolayer remains poorly elucidated. We used real-time cell visualization to describe the relationship between FA parameters and migration of immortalized epithelial keratinocytes (HaCaT) and lung carcinoma cells (A549) under inhibition or depletion of the FA proteins vinculin and FAK. To evaluate the relationship between FA morphology and cell migration, we used substrates of different elasticity in a wound healing assay. High FAK and vinculin mRNA expression, as well as largest FAs and maximal migration rate were described for cells on fibronectin, whereas cells plated on glass had minimal FA area and decelerated speed of migration into the wound. Both for normal and tumor cells, suppression of vinculin expression resulted in decreased FA size and fluorescence intensity, but had no effect on cell migration into the wound. Suppression of FAK expression or inhibition of FAK activity had no effect on FA size, but decreased FA lifetime and significantly slowed the rate of wound healing both for HaCaT and A549 cells. Our data indicates that FA lifetime, but not FA area is essential for epithelial cell migration within a monolayer. The effect of FAK kinase on the rate of cell migration within the monolayer makes FAK a promising target for antitumor therapy of lung adenocarcinoma.

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Sobre autores

E. Solomatina

Lomonosov Moscow State University; Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: aleena.saidova@gmail.com
Rússia, Moscow; Moscow

A. Kovaleva

Lomonosov Moscow State University; Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: aleena.saidova@gmail.com
Rússia, Moscow; Moscow

A. Tvorogova

Lomonosov Moscow State University

Email: aleena.saidova@gmail.com
Rússia, Moscow

I. Vorobyov

Lomonosov Moscow State University

Email: aleena.saidova@gmail.com
Rússia, Moscow

A. Saidova

Lomonosov Moscow State University; Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Autor responsável pela correspondência
Email: aleena.saidova@gmail.com
Rússia, Moscow; Moscow

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2. Fig. 1. PC morphology correlates with the rate of wound healing on substrates of different stiffness. a - PC morphology in A549 and HaCaT cells with stable vinculin-RFP expression, size cutoff 10 μm, C - glass, FN - fibronectin, photos of marginal cells in the monolayer are shown. b - Median PC area in A549 cells on substrates of different stiffness, the central horizontal line is the median. **** Significant difference from control, p < 0.001. c - Degree of experimental wound healing in % of the area at the first imaging frame for A549 cells on glass (1), poly-D-lysine (2), vitronectin (3) and fibronectin (4)

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3. Fig. 2. Normalised vinculin (a, c) and FAK (b, d) mRNA expression in A549 and HaCaT cells in control and after suppression of vinculin or FAK mRNA expression in the cells. All measurements were performed in triplicate and data were normalised for UBC and HPRT1 genes. Denotations: (1) control cells on glass, (2) control cells on fibronectin, (3) cells with miRNA to vinculin on glass, (4) cells with miRNA to vinculin on fibronectin, (5) cells with miRNA to FAK on glass, (6) cells with miRNA to FAK on fibronectin, (7) cells with miRNA to GFP. ** Significant difference from control, p < 0.01;*** Significant difference from control, p < 0.001

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4. Fig. 3. FA parameters and rate of wound healing by A549 cells after suppression of FAK or vinculin mRNA expression. a - FA morphology on glass and fibronectin in control and after vinculin knockdown (KD vin), size section 10 μm. b - Staining of fixed A549 cells with stable vinculin-RFP expression with antibodies to paxillin. Denotations: 1 - control, 2 - cells with miRNA to vinculin, 3 - cells with miRNA to FAK, I - staining with antibodies to vinculin, II - staining with antibodies to paxillin, III - superimposition. c - FA lifetime in A549 cells, bold horizontal lines represent medians. Designations: 1 - control cells on fibronectin, 2 - control cells on glass, 3 - cells with miRNA to vinculin on fibronectin, 4 - cells with miRNA to vinculin on glass, 5 - cells with miRNA to FAK on fibronectin, 6 - cells with miRNA to FAK on glass. ** Significant difference from control, p < 0.01. d - Morphology of experimental wound overgrowth by A549 cells on fibronectin and on glass in control (1) and after vinculin knockdown (2). e - Degree of overgrowth of the experimental wound in % of area at the first imaging frame for A549 cells on fibronectin in control (1), on fibronectin in cells with miRNA to vinculin (2), on glass in control (3) and on glass in cells with miRNA to vinculin (4). f - Degree of experimental wound overgrowth in % of area at the first imaging frame for A549 cells on fibronectin in control (1), on fibronectin in cells with miRNA to FAK(2), on glass in control (3) and on glass in cells with miRNA to FAK (4). g - Morphology of experimental wound overgrowth by A549 cells on fibronectin and on glass in control (1) and after FAK knockdown (2). The number of experiments for each wound overgrowth analysis is given in Table S3 of the Supplementary Material

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5. Fig. 4. FA parameters and rate of wound healing by HaCaT cells after suppression of FAK or vinculin mRNA expression. a - Morphology of FA on glass and fibronectin in control and after vinculin knockdown (KD vin), size section 10 μm. b - Staining of fixed HaCaT cells with stable expression of vinculin RFP with antibodies to paxillin, 1 - control, 2 - cells with miRNA to vinculin, 3 - cells with miRNA to FAK, I - staining with antibodies to vinculin, II - staining with antibodies to paxillin, III - overlay. c - Lifetime of FAs in HaCaT cells, bold horizontal lines represent medians. Designations: (1) control cells on fibronectin, (2) control cells on glass, (3) cells with miRNA to FAK on fibronectin, (4) cells with miRNA to FAK on glass. ** Significant difference from control, p < 0.01; **** significant difference from control, p < 0.0001. d - Morphology of experimental wound overgrowth by HaCaT cells on glass in control (1), after vinculin knockdown (2) and after FAK knockdown (3). e - Degree of experimental wound overgrowth in % of area at the first imaging frame for A549 cells on fibronectin in control (1), on fibronectin in cells with miRNA to vinculin (2), on glass in control (3) and on glass in cells with miRNA to vinculin (4). f - Degree of experimental wound overgrowth in % of area at the first imaging frame for A549 cells on fibronectin in control (1), on fibronectin in cells with miRNA to FAK (2), on glass in control (3) and on glass in cells with miRNA to FAK (4). g - Morphology of experimental wound overgrowth by A549 cells on fibronectin and on glass in control (1), after vinculin knockdown (2) and after FAK knockdown (3). The number of experiments for each wound healing assay is shown in Table S3 of the Supplementary Material

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6. Fig. 5. Overgrowth of experimental wound on fibronectin and glass. a - Overgrowth of experimental wound by A549 cells on fibronectin and glass in control (1) and after incubation with 1 μM PF-573228 (2). b - Overgrowth of model wound by HaCaT cells on fibronectin and glass in control (1) and after incubation with 1 μM PF-573228 (2). c - Degree of experimental wound overgrowth in % of area in the first imaging frame for A549 cells on fibronectin in control (1), on fibronectin in cells after incubation with 1 μM PF-573228 (2), on glass in control (3) and on glass in cells after incubation with 1 μM PF-573228 (4). d - Degree of experimental wound overgrowth in % of area at the first imaging frame for HaCaT cells on fibronectin in control (1), on fibronectin in cells after incubation with 1 μM PF-573228 (2), on glass in control (3) and on glass in cells after incubation with 1 μM PF-573228 (4)

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