K+ Channel Tetramerization Domain 5 (KCTD5) Protein Regulates Cell Migration, Focal Adhesion Dynamics and Spreading through Modulation of Ca2+ Signaling and Rac1 Activity
"> Figure 1
<p>Potassium Channel Tetramerization Domain 5 (KCTD5) regulates migration of B16-F10 cells. (<b>A</b>) Evaluation of KCTD5 levels by immunoblot in B16-F10 cells transfected with shRNA<sup>Scramble</sup>, shRNA<sup>KCTD5</sup># 1 or shRNA<sup>KCTD5</sup> #2 encoding plasmids. The KCTD5 levels were normalized to α-tubulin loading control. Graph represents the relative KCTD5 levels for each condition (mean ± SD; <span class="html-italic">n</span> = 3; One-way ANOVA followed by Tukey’s multiple comparisons test, ** <span class="html-italic">p</span> < 0.01). (<b>B</b>) Transwell Boyden chamber migration assays of B16-F10 cells transfected with shRNA<sup>Scramble</sup>, shRNA<sup>KCTD5</sup> #1 or shRNA<sup>KCTD5</sup> #2 encoding plasmids. Cells were stimulated with 10% <span class="html-italic">v</span>/<span class="html-italic">v</span> serum for 18 h. Scale bar = 500 μm. Graph represents the relative migration for each condition (mean ± SD; <span class="html-italic">n</span> = 5; One-way ANOVA followed Tukey’s multiple comparisons test, * <span class="html-italic">p</span> < 0.05). (<b>C</b>) Representative immunoblots showing KCTD5 levels in B16-F10<sup>Control</sup> and CRISPR/Cas9-based KCTD5 Knockout B16-F10 cells (B16-F10<span class="html-italic"><sup>kctd5-</sup></span><sup>/<span class="html-italic">-</span></sup>). α-tubulin was used as loading control. (<b>D</b>) Analysis of KCTD5 mRNA expression by RT-qPCR. The KCTD5 mRNA levels were normalized to β-Actin mRNA. Graph represents the relative KCTD5 mRNA levels for B16-F10<sup>Control</sup> and B16-F10<span class="html-italic"><sup>kctd5-</sup></span><sup>/<span class="html-italic">-</span></sup> cells (mean ± SD; <span class="html-italic">n</span> = 3; two-tailed unpaired Student’s <span class="html-italic">t</span>-test, * <span class="html-italic">p</span> < 0.05). (<b>E</b>) Transwell Boyden chamber migration assays of B16-F10<sup>Control</sup> and B16-F10<span class="html-italic"><sup>kctd5-</sup></span><sup>/<span class="html-italic">-</span></sup> cells. Cells were stimulated with 10% <span class="html-italic">v</span>/<span class="html-italic">v</span> serum for 18 h. Scale bar = 500 μm. Graph represents the relative migration for each condition (mean ± SD; <span class="html-italic">n</span> = 3; two-tailed unpaired Student’s <span class="html-italic">t</span>-test, * <span class="html-italic">p</span> < 0.05). (<b>F</b>) Representative immunoblots showing levels of EGFP, EGFP-KCTD5 and endogenous KCTD5 in EGFP- or EGFP-KCTD5-transfected B16-F10<sup>Control</sup> and B16-F10<span class="html-italic"><sup>kctd5-</sup></span><sup>/<span class="html-italic">-</span></sup> cells. α-tubulin was used as loading control. (<b>G</b>) Transwell Boyden chamber migration assays of B16-F10<sup>Control</sup> and B16-F10<span class="html-italic"><sup>kctd5-</sup></span><sup>/<span class="html-italic">-</span></sup> cells transfected with EGFP or EGFP-KCTD5 encoding plasmids. Cells were stimulated with 10% <span class="html-italic">v</span>/<span class="html-italic">v</span> serum for 18 h. Scale bar = 500 μm. Graph represents the relative migration for each condition (mean ± SD; <span class="html-italic">n</span> = 3; Two-way ANOVA followed by Sidak’s multiple comparisons test, ns = not significant, * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01).</p> "> Figure 2
<p>KCTD5 regulates spreading of B16-F10 cells. (<b>A</b>) Representative images of shRNA<sup>Scramble</sup>-, shRNA<sup>KCTD5</sup> #1- or shRNA<sup>KCTD5</sup> #2-transfected B16-F10 cells incubated for 45 or 90 min on fibronectin-coated coverslips. Cells were fixed and stained for F-actin with Alexa-555 phalloidin (red). Focal adhesions were labeled with mouse mAb anti-vinculin (cyan). EGFP positive cells were analyzed. Scale bar = 0.25 µm. (<b>B</b>) Graph representing the cell area (µm<sup>2</sup>) for each condition (mean ± SD; <span class="html-italic">n</span> = 4; Two-way ANOVA followed by Tukey’s and Sidak’s multiple comparisons tests, ** <span class="html-italic">p</span> < 0.01, **** <span class="html-italic">p</span> < 0.0001). (<b>C</b>) Graph representing the percentage of lamellipodial-like cells for each condition (mean ± SD; <span class="html-italic">n</span> = 4; Two-way ANOVA followed by Tukey’s and Sidak’s multiple comparisons tests, * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001). (<b>D</b>) Representative images of B16-F10<sup>Control</sup> and B16-F10<span class="html-italic"><sup>kctd5-/-</sup></span> cells incubated for 45 or 90 min on fibronectin-coated coverslips. Cells were fixed and stained for F-actin with Alexa-488 phalloidin (green) and for nuclei with Hoechst reagent (blue). Arrows show lamellipodial-like cells. White boxes show the magnification area. Scale bar = 0.20 µm. (<b>E</b>) Graph representing the cell area (µm<sup>2</sup>) for each condition (mean ± SD; <span class="html-italic">n</span> = 4; Two-way ANOVA followed by Sidak’s multiple comparisons test, ** <span class="html-italic">p</span> < 0.01, **** <span class="html-italic">p</span> < 0.0001). (<b>F</b>) Graph representing the percentage of lamellipodial-like cells for each condition (mean ± SD; <span class="html-italic">n</span> = 4; Two-way ANOVA followed by Sidak’s multiple comparisons test, ** <span class="html-italic">p</span> < 0.01). (<b>G</b>) Representative images of EGFP- or EGFP-KCTD5-transfected B16-F10<sup>Control</sup> and B16-F10<span class="html-italic"><sup>kctd5-/-</sup></span> cells incubated for 45 min on fibronectin-coated coverslips. Cells were fixed and stained for F-actin with Alexa-555 phalloidin (red). EGFP positive cells were analyzed. Scale bar = 0.25 µm. (<b>H</b>) Graph representing the cell area (µm<sup>2</sup>) for each condition (mean ± SD; <span class="html-italic">n</span> = 4; Two-way ANOVA followed by Sidak’s multiple comparisons test, **** <span class="html-italic">p</span> < 0.0001). (<b>I</b>) Graph representing the percentage of lamellipodial-like cells for each condition (mean ± SD; <span class="html-italic">n</span> = 4; Two-way ANOVA followed by Sidak’s multiple comparisons test, **** <span class="html-italic">p</span> < 0.001).</p> "> Figure 3
<p>KCTD5 regulates cell spreading and migration through Rac1. (<b>A</b>) Representative images of B16-F10<sup>Control</sup> and B16-F10<span class="html-italic"><sup>kctd5-</sup></span><sup>/<span class="html-italic">-</span></sup> cells transfected with empty vector (Mock), myc-Rac1-Q61L (constitutively active) or myc-Rac1-T17N (dominant-negative) encoding plasmids. Cells were seeded on fibronectin-coated coverslips for 45 min, fixed and stained for F-actin with Alexa-488 phalloidin (green). Rac1 mutants-transfected cells were labeled with mouse mAb anti-c-myc (red). Arrows show lamellipodial-like cells. White boxes show magnification area. Scale bar = 0.20 µm. (<b>B</b>) Graph representing the cell area (µm<sup>2</sup>) for each condition (mean ± SD; <span class="html-italic">n</span> = 4; Two-way ANOVA followed by Tukey’s and Sidak’s multiple comparisons tests, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001, **** <span class="html-italic">p</span> < 0.0001). (<b>C</b>) Graph representing the percentage of lamellipodial-like cells for each condition (mean ± SD; <span class="html-italic">n</span> = 4; Two-way ANOVA followed by Tukey’s and Sidak’s multiple comparisons tests, **** <span class="html-italic">p</span> < 0.0001). (<b>D</b>) Representative immunoblot showing levels of myc-Rac1-Q61L (constitutively active), myc-Rac1-T17N (dominant-negative) and KCTD5 in B16-F10<sup>Control</sup> and B16-F10<span class="html-italic"><sup>kctd5-</sup></span><sup>/<span class="html-italic">-</span></sup> cells transfected with empty vector (Mock), myc-Rac1-Q61L or myc-Rac1-T17N encoding plasmids. α-tubulin was used as loading control. (<b>E</b>) Transwell Boyden chamber migration assays of B16-F10<sup>Control</sup> and B16-F10<span class="html-italic"><sup>kctd5-</sup></span><sup>/<span class="html-italic">-</span></sup> cells transfected with empty vector (Mock) or myc-Rac1-Q61L encoding plasmid. Cells were stimulated with 10 % <span class="html-italic">v</span>/<span class="html-italic">v</span> serum for 18 h. Scale bar = 500 µm. Graph represents the relative migration for each condition (mean ± SD; <span class="html-italic">n</span> = 3; Two-way ANOVA followed by Sidak’s multiple comparisons tests, * <span class="html-italic">p</span> < 0.05).</p> "> Figure 4
<p>KCTD5 regulates focal adhesion size of B16-F10 cells. (<b>A</b>) Representative images of B16-F10 cells transfected with shRNA<sup>Scramble</sup>, shRNA<sup>KCTD5</sup> #1 or shRNA<sup>KCTD5</sup> #2 encoding plasmids. Focal adhesions were labeled with mouse mAb anti-vinculin (red). Focal adhesions of EGFP positive cells were analyzed. Scale bar = 25 μm. (<b>B</b>) Graph representing the Average of focal adhesion area per cell (µm<sup>2</sup>) for each condition (mean ± SD; <span class="html-italic">n</span> = 5; One-way ANOVA followed by Tukey’s multiple comparisons test, **** <span class="html-italic">p</span> < 0.0001). (<b>C</b>) Graph representing the Number of focal adhesions per cell for each condition (mean ± SD; <span class="html-italic">n</span> = 5; One-way ANOVA followed by Tukey’s multiple comparisons test, ns = not significant). (<b>D</b>) Representative images of B16-F10<sup>Control</sup> and CRISPR/Cas9-based KCTD5 Knockout (B16-F10<span class="html-italic"><sup>kctd5-</sup></span><sup>/<span class="html-italic">-</span></sup>) cells immunostained against the focal adhesion protein vinculin (red). F-actin was visualized by Alexa 488-phalloidin stain. Scale bar = 25 μm. (<b>E</b>) Graph representing the Average of focal adhesion area per cell (µm<sup>2</sup>) for each condition (mean ± SD; <span class="html-italic">n</span> = 5; two-tailed unpaired Student’s <span class="html-italic">t</span>-test, **** <span class="html-italic">p</span> < 0.0001). (<b>F</b>) Graph representing the number of focal adhesions per cell for each condition (mean ± SD; <span class="html-italic">n</span> = 5; two-tailed unpaired Student’s <span class="html-italic">t</span>-test, ns = not significant). (<b>G</b>) Representative images showing tracked focal adhesions (red arrowhead and green mark) and time (in minutes) from B16-F10 cells co-transfected with mCherry-paxillin construct and shRNA<sup>Scramble</sup> or shRNA<sup>KCTD5</sup> #1 encoding plasmids. Serum-starved cells were stimulated with 10% <span class="html-italic">v</span>/<span class="html-italic">v</span> Fetal Bovine Serum (FBS) to induce the focal adhesion assembly/disassembly. Focal adhesions were tracked by mCherry-paxillin fluorescence. Focal adhesion dynamics were obtained by live-cell time-lapse recording. (<b>H</b>) Graph representing the average assembly rate (mean ± SD; <span class="html-italic">n</span> = 3; two-tailed unpaired Student’s <span class="html-italic">t</span>-test, ns = not significant). (<b>I</b>) Graph representing the average disassembly rate (mean ± SD; <span class="html-italic">n</span> = 3; two-tailed unpaired Student’s <span class="html-italic">t</span>-test, ** <span class="html-italic">p</span> < 0.01).</p> "> Figure 5
<p>KCTD5 promotes serum-induced Ca<sup>2+</sup> signals in B16-F10 cells. (<b>A</b>) Time courses for normalized fluorescence in B16-F10 cells transfected with shRNA<sup>Scramble</sup>, shRNA<sup>KCTD5</sup> #1 or shRNA<sup>KCTD5</sup> #2 encoding plasmids. Cells were loaded with 5 µM Fura-2-AM probe and Ca<sup>2+</sup> peak was induced by 10% <span class="html-italic">v</span>/<span class="html-italic">v</span> FBS in 2.5 mM CaCl<sub>2</sub> Krebs medium. Each point corresponds to the mean ± SEM. (<b>B</b>) Graph representing the mean ± SEM of ΔF<sub>maximum</sub>/F<sub>0</sub> for each condition (<span class="html-italic">n</span> = 7; One-way ANOVA followed by Tukey’s multiple comparisons test, ** <span class="html-italic">p</span> < 0.01, **** <span class="html-italic">p</span> < 0.0001). (<b>C</b>) Time courses for normalized fluorescence in B16-F10<sup>Control</sup> cells transfected with EGFP or EGFP-KCTD5 encoding plasmids. Cells were loaded with 5 µM Fura-2-AM probe and Ca<sup>2+</sup> peak was induced by 10% <span class="html-italic">v</span>/<span class="html-italic">v</span> FBS in 2 mM CaCl<sub>2</sub> Krebs medium. Each point corresponds to the mean ± SEM. (<b>D</b>) Graph representing the mean ± SEM of ΔF<sub>maximum</sub>/F<sub>0</sub> for each condition (<span class="html-italic">n</span> = 5; two-tailed unpaired Student’s <span class="html-italic">t</span>-test, **** <span class="html-italic">p</span> < 0.0001). (<b>E</b>) Time courses for normalized fluorescence in B16-F10<sup>Control</sup> cells expressing EGFP and B16-F10<span class="html-italic"><sup>kctd5-</sup></span><sup>/<span class="html-italic">-</span></sup> cells expressing EGFP or EGFP-KCTD5 construct. Cells were loaded with 5 µM Fura-2-AM probe and Ca<sup>2+</sup> peak was induced by 10% <span class="html-italic">v</span>/<span class="html-italic">v</span> FBS in 2 mM CaCl<sub>2</sub> Krebs medium. Each point corresponds to the mean ± SEM. (<b>F</b>) Graph representing the mean ± SEM of ΔF<sub>maximum</sub>/F<sub>0</sub> for each condition (<span class="html-italic">n</span> = 5; One-way ANOVA followed by Tukey’s multiple comparisons test, * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01).</p> "> Figure 6
<p>Ca<sup>2+</sup> mediates the KCTD5-promoted focal adhesion size regulation. (<b>A</b>) Representative images of B16-F10 transfected with shRNA<sup>Scramble</sup> or shRNA<sup>KCTD5</sup> #1 encoding plasmids. Cells were serum-starved for 4 h and then were treated with 1 μM ionomycin in a 2 mM CaCl<sub>2</sub> Krebs medium for 20 min. DMSO was used as a vehicle control. Focal adhesions were labeled with mouse mAb anti-vinculin (red). Focal adhesions of EGFP positive cells were analyzed. Scale bar = 25 μm. (<b>B</b>) Graph representing the Average of focal adhesion area per cell (µm<sup>2</sup>) for each condition (mean ± SD; <span class="html-italic">n</span> = 4; Two-way ANOVA followed by Sidak’s multiple comparisons test, **** <span class="html-italic">p</span> < 0.0001). (<b>C</b>) Graph representing the Number of focal adhesions per cell for each condition (mean ± SD; <span class="html-italic">n</span> = 5; Two-way ANOVA followed by Sidak’s multiple comparisons test, * <span class="html-italic">p</span> < 0.05). (<b>D</b>) Representative images of B16-F10 transfected with shRNA<sup>Scramble</sup> or shRNA<sup>KCTD5</sup> #1 encoding plasmids. Cells were serum-starved for 4 h and then were treated with 1 μM ionomycin in a Ca<sup>2+</sup>-free (0 Ca<sup>2+</sup>) or a Ca<sup>2+</sup>-containing (2 mM Ca<sup>2+</sup>) Krebs medium for 20 min. Focal adhesions were labeled with mouse mAb anti-vinculin (red). Focal adhesions of EGFP positive cells were analyzed. Scale bar = 25 μm. (<b>E</b>) Graph representing the Average of focal adhesion area per cell (µm<sup>2</sup>) for each condition (mean ± SD; <span class="html-italic">n</span> = 4; Two-way ANOVA followed by Sidak’s multiple comparisons test, ** <span class="html-italic">p</span> < 0.01, **** <span class="html-italic">p</span> < 0.0001). (<b>F</b>) Graph representing the Number of focal adhesions per cell for each condition (mean ± SD; <span class="html-italic">n</span> = 4; Two-way ANOVA followed by Sidak’s multiple comparisons test, *** <span class="html-italic">p</span> < 0.001).</p> "> Figure 7
<p>Proposed model. KCTD5 regulates cell migration by two paths: Ca<sup>2+</sup> signaling and Rac1 activity. KCTD5 promotes the serum-induced Ca<sup>2+</sup> entry and/or Ca<sup>2+</sup> release from intracellular storage, leading to a higher global Ca<sup>2+</sup> signal. This global Ca<sup>2+</sup> rise inhibits the focal adhesion turnover. On the other hand, KCTD5 inhibits the Rac1 activity, leading to a decreased lamellipodia formation. All these effects suggest that KCTD5 plays a role as a negative modulator for B16-F10 cells migration.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Reagents
2.2. Plasmids
2.3. Antibodies
2.4. Cell Culture and Transfection
2.5. KCTD5 Knockout B16-F10 (B16-F10kctd5-/-) and Control B16-F10 (B16-F10Control) Cells Generation
2.6. Immunoblot Analyses
2.7. mRNA Expression Analyses by RT-qPCR
2.8. Boyden Chamber Transwell Migration Assays
2.9. Spreading Assay
2.10. Focal Adhesion Analyses
2.11. Focal Adhesions Dynamics Analyses
2.12. Intracellular Calcium Measurements
2.13. Statistical Analyses
3. Results
3.1. KCTD5 Depletion Increases Cell Migration of B16-F10 Cells
3.2. KCTD5 Depletion Enhances the Spreading of B16-F10 Cells
3.3. Cell Spreading and Migration Promoted by KCTD5-Depletion Is Dependent on Rac1 Activity
3.4. KCTD5 Depletion Affects Focal Adhesion Dynamics of B16-F10 Cells
3.5. KCTD5 Depletion Impacts in Serum-Induced Ca2+ Signaling
3.6. Serum-Induced Ca2+ Rise Is Necessary for KCTD5-Mediated Focal Adhesion Regulation
4. Discussion
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Antibody | Isotype | Dilution | Final µg/mL | Source | Type | Catalog# | RRID | Purifi- Cation |
---|---|---|---|---|---|---|---|---|
Mouse anti-KCTD5 | IgG2b | 1:1000 (IB) | 1 | Origene | mAb | TA501035 | AB_11140321 | Asc |
Mouse anti-tubulin | IgG1 | 1:5000 (IB) | 1 | Sigma-Aldrich | mAb | T5168 | AB_477579 | Asc |
Mouse anti-GFP | IgG2a | 1:2000 (IB) | 0.1 | Santa Cruz Biotechnology, Inc. | mAb | sc-9996 | AB_627695 | NA |
Mouse anti-vinculin | IgG1 | 1:200 (IF) | NA | Sigma-Aldrich | mAb | V4505 | AB_477617 | Asc |
Mouse anti-c-Myc | IgG1 | 1:200 (IF) | 10 | Sigma-Aldrich | mAb | M4439 | AB_439694 | AP |
Mouse anti-c-Myc | IgG1 | 1:2000 (IB) | 1 | Sigma-Aldrich | mAb | M4439 | AB_439694 | AP |
Mouse anti-Rac1 | IgG | 1:500 (IB) | 1 | Cytoskeleton, Inc. | mAb | ARC03 | AB_2721173 | NA |
Alexa Fluor 555 conjugated goat anti-Mouse IgG1 | IgG | 1:1000 | 2 | Thermo Fisher Scientific | pAb | A-21127 | AB_2535769 | AP |
Alexa Fluor 647 conjugated goat anti-Mouse IgG1 | IgG | 1:1000 | 2 | Thermo Fisher Scientific | pAb | A-21240 | AB_2535809 | AP |
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Canales, J.; Cruz, P.; Díaz, N.; Riquelme, D.; Leiva-Salcedo, E.; Cerda, O. K+ Channel Tetramerization Domain 5 (KCTD5) Protein Regulates Cell Migration, Focal Adhesion Dynamics and Spreading through Modulation of Ca2+ Signaling and Rac1 Activity. Cells 2020, 9, 2273. https://doi.org/10.3390/cells9102273
Canales J, Cruz P, Díaz N, Riquelme D, Leiva-Salcedo E, Cerda O. K+ Channel Tetramerization Domain 5 (KCTD5) Protein Regulates Cell Migration, Focal Adhesion Dynamics and Spreading through Modulation of Ca2+ Signaling and Rac1 Activity. Cells. 2020; 9(10):2273. https://doi.org/10.3390/cells9102273
Chicago/Turabian StyleCanales, Jimena, Pablo Cruz, Nicolás Díaz, Denise Riquelme, Elías Leiva-Salcedo, and Oscar Cerda. 2020. "K+ Channel Tetramerization Domain 5 (KCTD5) Protein Regulates Cell Migration, Focal Adhesion Dynamics and Spreading through Modulation of Ca2+ Signaling and Rac1 Activity" Cells 9, no. 10: 2273. https://doi.org/10.3390/cells9102273