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. 2020 Mar 3;8:125.
doi: 10.3389/fcell.2020.00125. eCollection 2020.

KANK2 Links αVβ5 Focal Adhesions to Microtubules and Regulates Sensitivity to Microtubule Poisons and Cell Migration

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Free PMC article

KANK2 Links αVβ5 Focal Adhesions to Microtubules and Regulates Sensitivity to Microtubule Poisons and Cell Migration

Mladen Paradžik et al. Front Cell Dev Biol. .
Free PMC article

Abstract

Integrins are heterodimeric glycoproteins that bind cells to extracellular matrix. Upon integrin clustering, multimolecular integrin adhesion complexes (IACs) are formed, creating links to the cell cytoskeleton. We have previously observed decreased cell migration and increased sensitivity to microtubule (MT) poisons, paclitaxel and vincristine, in the melanoma cell line MDA-MB-435S upon transfection with integrin αV-specific siRNA, suggesting a link between adhesion and drug sensitivity. To elucidate the underlying mechanism, we determined αV-dependent changes in IAC composition. Using mass spectrometry (MS)-based proteomics, we analyzed the components of isolated IACs of MDA-MB-435S cells and two MDA-MB-435S-derived integrin αV-specific shRNA-expressing cell clones with decreased expression of integrin αV. MS analysis showed that cells preferentially use integrin αVβ5 for the formation of IACs. The differential analysis between MDA-MB-435S cells and clones with decreased expression of integrin αV identified key components of integrin αVβ5 adhesion complexes as talins 1 and 2, α-actinins 1 and 4, filamins A and B, plectin and vinculin. The data also revealed decreased levels of several components of the cortical microtubule stabilization complex, which recruits MTs to adhesion sites (notably liprins α and β, ELKS, LL5β, MACF1, KANK1, and KANK2), following αV knockdown. KANK2 knockdown in MDA-MB-435S cells mimicked the effect of integrin αV knockdown and resulted in increased sensitivity to MT poisons and decreased migration. Taken together, we conclude that KANK2 is a key molecule linking integrin αVβ5 IACs to MTs, and enabling the actin-MT crosstalk that is important for both sensitivity to MT poisons and cell migration.

Keywords: KANK2; adhesome; antitumor drug resistance; cell migration; cortical microtubule stabilizing complex; integrin αVβ5.

Figures

FIGURE 1
FIGURE 1
Characterization of the MDA-MB-435S cell model. Surface expression of integrin subunit αV, integrin heterodimers αVβ3 and αVβ5, and integrin subunit β1 in melanoma cell line MDA-MB-435S and clones 2αV and 3αV obtained by stable transfection with integrin αV-specific shRNA. Cells were detached by EDTA and analyzed by flow cytometry upon incubation with antibodies against integrin subunit αV, β1, or integrin heterodimers αVβ3 or αVβ5 (black histogram), and isotype-matched antibody as a negative control (gray histogram), followed by rabbit FITC-conjugated-anti-mouse antibody (upper panel). Results from upper panel were presented as comparisons of MFIs within the cell model (lower panel). Representative data of three independent experiments yielding similar results are shown.
FIGURE 2
FIGURE 2
Effect of integrin αV knockdown on cell sensitivity to antitumor drugs, PTX-induced apoptosis, cell proliferation, growth, spreading and migration. (A) Clones 2αV and 3αV demonstrate decreased sensitivity to cDDP and increased sensitivity to VCR and PTX as compared to parental MDA-MB-435S cells. Cells were seeded in 96-well plates and 24 h later treated with different concentrations of cDDP, VCR, and PTX. Cytotoxicity was measured by MTT assay. Results presented are representative of three independent experiments with similar results ± SD. Data were analyzed by unpaired Student’s t-test. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. (B) Clones 2αV and 3αV upon PTX treatment demonstrate increased apoptosis as compared to MDA-MB-435S cells. Cells were treated with 0.004 μg/mL of PTX for 72 h, harvested for Annexin V/PI staining and analyzed by flow cytometry to discriminate between live (lower left quadrant), apoptotic and/or necrotic cells (right quadrants). The representative data of three independent experiments yielding similar results are shown. (C) Cell proliferation in MDA-MB-435S cells and clones 2αV and 3αV. Cell proliferation was measured using ClickIT EdU assay. DNA synthesis was measured upon 2 h cell growth in medium supplemented with EdU and the amount of incorporated EdU was measured by flow cytometry. Comparison of average percentage of EdU + cells from three different experiments are shown. (D) Growth curve of MDA-MB-435S cells and clones 2αV and 3αV. Cells were seeded in 10 cm culture plates and counted on days 1, 2, and 3. The results presented are representative of three independent experiments with similar results. (E) Cell spreading of MDA-MB-435S cells and clones 2αV and 3αV. Live cell imaging was performed during 18–20 h upon seeding and cell spreading was compared using time lapse IRM images (30 min–12 h). Scale bar = 10 μm. (F) Decreased migration of clones 2αV and 3αV as compared to MDA-MB-435S cells. Serum starved (24 h) cells were seeded in Transwell cell culture inserts and left to migrate for 22 h toward serum. Cells on the underside of the inserts were stained with crystal violet, photographed, and counted. Scale bar = 100 μm. Averages of five microscope fields of three independently performed experiments ± SD are shown (n = 3). Data were analyzed by one-way ANOVA with Dunnett’s multiple Comparison. ***P < 0.001.
FIGURE 3
FIGURE 3
Clones 2αV and 3αV have decreased number and size of FAs and decreased amount of stress fibers per cell as compared to MDA-MB-435S cells. (A) Clones 2αV and 3αV show decreased expression of the integrin subunit αV and the FA marker, paxillin. Forty-eight hours after seeding on coverslips, cells were fixed, permeabilized and stained with antibody against integrin αV or paxillin followed by Alexa-Fluor 488-conjugated antibody (green). F-actin staining (red) was performed in all samples, and nuclei were stained with DAPI (blue). Analysis was performed using TCS SP8 Leica. Scale bar = 10 μm. (B) Quantification of αV and paxillin, FA size and % of stress fibers per cell. Data presented as histograms or scatter plots (FA size) represent measurements of > 50 cells and are plotted as mean ± SD (n = 3). Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison. ***P < 0.001.
FIGURE 4
FIGURE 4
Mass spectrometry analysis of IACs isolated from MDA-MB-435S cells and clones 2αV and 3αV. (A) Protein–protein interaction network of components identified by MS in IACs isolated form MDA-MB-435S cells. Shapes represent identified proteins and are labeled with gene symbols, arranged and colored according to their functional group as indicated (CMSC). In case of multiple functional terms assigned for each protein, the molecular function assigned by both databases has been chosen for interpretation of results. (B) Total identified IAC proteins in MDA-MB-435S cells (number of spectral counts ≥ 4, FDR < 5%, probability for protein identification ≥ 99.9%) were annotated using David GO database. To determine the sample enrichment, P-values related to GO terms of cellular components (GOTERM_CC_DIRECT), were used. Analysis of gene ontology terms was performed using REViGO tool. Statistically significant GO terms (P = 0.05) were presented from highest P-value (bottom) to the lowest (top). (C) Volcano plot of MDA-MB-435S versus 2αV (red) and 3αV (black). To determine the significantly changed proteins −Log (FDR) ≥ 1 and fold change ≥ 1.5 were used. Upper left quadrant – proteins detected at lower level of spectra, upper right quadrant – proteins detected at higher level of spectra. (D) Venn diagram – proteins with higher (green arrow) and lower (red arrow) level of spectra detected in clones 2αV and 3αV versus MDA-MB-435S cells. Proteins found in both clones with changed abundances were showed in the intersected white area of the diagram. (E) DAVID GO analysis of proteins detected with higher (green arrow) and lower (red arrow) abundances. Statistically significant GO terms (P = 0.05; dashed line) were presented from highest P-value (bottom) to the lowest (top).
FIGURE 5
FIGURE 5
MS data validation in MDA-MB-435S cells and clones 2αV and 3αV. (A) Clones 2αV and 3αV show decreased expression of vinculin, talin 1/2 and α-actinin 1 as compared to MDA-MB-435S cells. Forty-eight hours after seeding on coverslips, cells were fixed, permeabilized, incubated with antibodies against vinculin, talin 1/2 or α-actinin 1 antibody, followed by Alexa-Fluor 488-conjugated antibody (green). F-actin staining (red) was performed in all samples, and nuclei were stained with DAPI (blue). Analysis was performed using TCS SP8 Leica. Scale bar = 10 μm. (B) Quantification data of results in (A) presented as histograms (FA number) and scatter plots (FA or α-actinin 1 size) represent measurements of > 50 cells and are plotted as mean ± SD (n = 3). Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison. *P < 0.05; **P < 0.01; ***P < 0.001. (C) WB analysis of IAC proteins from clones 2αV and 3αV and MDA-MB-435S cells. Forty-eight hours after seeding, IACs were isolated and WB analysis was performed. The results presented are representative of three independent experiments yielding similar results.
FIGURE 6
FIGURE 6
Characterization of αVβ5-associated adhesion complexes. (A) The most abundant integrin subunits found in IACs. Dataset consists of at least two different experiments. Average spectral count number shown. (B) Clones with integrin subunit αV knockdown show decreased expression of integrin αVβ5. Forty-eight hours after seeding on coverslips, cells were fixed, permeabilized, and stained with anti-αVβ5 antibody, followed by Alexa-Fluor 488-conjugated antibody (green). F-actin staining (red) was performed, and nuclei were stained with DAPI (blue). Analysis was performed using TCS SP8 Leica. Scale bar = 10 μm. (C) Quantification data of results in (B) presented as histogram (FA number) and scatter plot (FA size) represent measurements of > 50 cells and are plotted as mean ± SD (n = 2). Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison. ***P < 0.001. (D) Identification of reticular adhesion structures in MDA-MB-435S cells and clone 2αV. Forty-eight hours after seeding on coverslips, cells were fixed, permeabilized, and stained for anti-αVβ5 followed by Alexa-Fluor 488-conjugated antibody (green), and anti-vinculin followed by Alexa-Fluor 647-conjugated antibody (purple). F-actin staining (red) was performed, nuclei were stained with DAPI (blue) and IRM images were taken. Analysis was performed using TCS SP8 Leica. Scale bar = 10 μm.
FIGURE 7
FIGURE 7
KANK2 knockdown in MDA-MB-435S cells increases sensitivity to MT poisons and decreases migration mimicking integrin αV knockdown. (A) Average MS data of number of spectra specific for KANK2 in MDA-MB-435S cell model. Dataset consists of at least two different experiments. (B) KANK2 is present in lower amount in IACs of clones 2αV and 3αV. WB analysis of KANK2 in isolated IAC proteins from clones 2αV and 3αV and MDA-MB-435S cells. Forty-eight hours after seeding, IACs were isolated and WB analysis was performed. The results presented are representative of two independent experiments yielding similar results. (C) Knockdown of KANK2 in MDA-MB-435S cells. WB analysis of KANK2 from MDA-MB-435S cells transfected with either control (si(-)) or KANK2-specific siRNA (si(KANK2)). Forty-eight hours after transfection total cell lysates were collected and WB analysis was performed. The results presented are representative of two independent experiments yielding similar results. (D) MDA-MB-435S cells upon KANK2 knockdown demonstrate increased sensitivity to VCR and PTX as compared to MDA-MB-435S cells transfected with control siRNA. Twenty-four hours upon transfection, cells were seeded in 96-well plates and 24 h later treated with different concentrations of VCR and PTX. Cytotoxicity was measured by MTT assay. Results presented are representative of three independent experiments with similar results ± SD. Data were analyzed by unpaired Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001. (E) KANK2 knockdown decreases migration in MDA-MB-435S cells. Serum starved (24 h) cells, transfected previously with either control or KANK2-specific siRNA were seeded in Transwell cell culture inserts and left to migrate for 22 h toward serum. Cells on the underside of the inserts were stained with crystal violet, photographed, and counted. Scale bar = 100 μm. Averages of five microscope fields of three independently performed experiments ± SD are shown (n = 3). Data were analyzed by one-way ANOVA with Dunnett’s multiple Comparison. ***P < 0.001. (F) KANK2 knockdown in MDA-MB-435S cells does not alter cell size or amount of stress fibers but slightly alters appearance of MTs. Forty-eight hours after transfection of MDA-MB-435S cells with KANK2-specific or control siRNA cells were fixed, permeabilized, and stained with anti-KANK2 antibody, followed by Alexa-Fluor 488-conjugated antibody (green). The α-tubulin or F-actin staining (red) was performed, nuclei were stained with DAPI (blue) and IRM images were taken. Analysis was performed using TCS SP8 Leica. Scale bar = 10 μm. Quantification data of results are presented as histograms represent measurements of > 50 cells and are plotted as mean ± SD (n = 2). Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison. ***P < 0.001.

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