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TTBK2 with EB1/3 regulates microtubule dynamics in migrating cells through KIF2A phosphorylation.

Watanabe T, Kakeno M, Matsui T, Sugiyama I, Arimura N, Matsuzawa K, Shirahige A, Ishidate F, Nishioka T, Taya S, Hoshino M, Kaibuchi K - J. Cell Biol. (2015)

Bottom Line: TTBK2 depletion reduced MT lifetime (facilitated shrinkage and suppressed rescue) and impaired HeLa cell migration, and these phenotypes were partially restored by KIF2A co-depletion.Expression of nonphosphorylatable KIF2A, but not wild-type KIF2A, reduced MT lifetime and slowed down the cell migration.These findings indicate that TTBK2 with EB1/3 phosphorylates KIF2A and antagonizes KIF2A-induced depolymerization at MT plus ends for cell migration.

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Affiliation: Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, Showa, Nagoya 466-8550, Japan.

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TTBK2 regulates cell migration via KIF2A. (A) Still images during wound healing of HeLa cells transfected with control or TTBK2 siRNA. TTBK2 depletion inhibited the migration of HeLa cells. (B) Migratory tracks of representative control, TTBK2-depleted, KIF2A-depleted, or TTBK2- and KIF2A-depleted cells. (C) Three parameters (migration distance, velocity, and directionality) were measured. For the control, TTBK2 depletion, and TTBK2/KIF2A double depletion, ≥150 cells from more than four independent experiments were analyzed. For KIF2A depletion or rescue with TTBK2-WT, ≥100 cells or ≥60 cells from three independent experiments were analyzed, respectively. ***, P < 0.001 (one-way ANOVA, Tukey’s HSD). (D) MT organization in migrating HeLa cells during wound healing. The boxes in the top panels are enlarged in the bottom panels. MT density at the cell front was measured as the mean intensity in the region indicated. TTBK2 depletion decreased MT intensity at the cell front. The decrease in MT intensity was reversed by co-depletion of KIF2A. Single-plane images focused on the cell periphery were used for the analysis. The fluorescence intensity was quantified in >20 cells for each condition and is shown as a ratio to the control cell value. The data represent the mean ± SD of three independent experiments (total of >60 cells for each condition). ***, P < 0.001 (one-way ANOVA, Tukey’s HSD). (E) The effects of TTBK2 depletion on the migration of cerebellar granule neurons in vivo. Granule neurons expressing control shRNA migrated toward the IGL through the ML, but neurons expressing TTBK2 shRNA exhibited only minimal migration. The graph shows the quantitative results. Error bars indicate the SEM. ***, P < 0.001 (one-way ANOVA). The data used for the statistical analysis were obtained from 12 slices of three brains (>300 cells). Bars: (A and B) 100 µm; (D) 10 µm; (D, magnification) 5 µm; (E) 50 µm.
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fig7: TTBK2 regulates cell migration via KIF2A. (A) Still images during wound healing of HeLa cells transfected with control or TTBK2 siRNA. TTBK2 depletion inhibited the migration of HeLa cells. (B) Migratory tracks of representative control, TTBK2-depleted, KIF2A-depleted, or TTBK2- and KIF2A-depleted cells. (C) Three parameters (migration distance, velocity, and directionality) were measured. For the control, TTBK2 depletion, and TTBK2/KIF2A double depletion, ≥150 cells from more than four independent experiments were analyzed. For KIF2A depletion or rescue with TTBK2-WT, ≥100 cells or ≥60 cells from three independent experiments were analyzed, respectively. ***, P < 0.001 (one-way ANOVA, Tukey’s HSD). (D) MT organization in migrating HeLa cells during wound healing. The boxes in the top panels are enlarged in the bottom panels. MT density at the cell front was measured as the mean intensity in the region indicated. TTBK2 depletion decreased MT intensity at the cell front. The decrease in MT intensity was reversed by co-depletion of KIF2A. Single-plane images focused on the cell periphery were used for the analysis. The fluorescence intensity was quantified in >20 cells for each condition and is shown as a ratio to the control cell value. The data represent the mean ± SD of three independent experiments (total of >60 cells for each condition). ***, P < 0.001 (one-way ANOVA, Tukey’s HSD). (E) The effects of TTBK2 depletion on the migration of cerebellar granule neurons in vivo. Granule neurons expressing control shRNA migrated toward the IGL through the ML, but neurons expressing TTBK2 shRNA exhibited only minimal migration. The graph shows the quantitative results. Error bars indicate the SEM. ***, P < 0.001 (one-way ANOVA). The data used for the statistical analysis were obtained from 12 slices of three brains (>300 cells). Bars: (A and B) 100 µm; (D) 10 µm; (D, magnification) 5 µm; (E) 50 µm.

Mentions: Finally, we examined whether TTBK2 regulates cell migration via KIF2A because appropriate MT dynamics and integrity are required for cell migration (Watanabe et al., 2005; Kuijpers and Hoogenraad, 2011; Etienne-Manneville, 2013). We performed wound healing assays using HeLa cells. The control HeLa cells migrated toward the gap during wound healing, but TTBK2 depletion inhibited this cell migration (Fig. 7 A). Tracking of each cell demonstrated the directional and persistent migration of control cells, whereas TTBK2-depleted cells migrated less efficiently, including a loss of directionality toward the wound (Fig. 7 B). When quantifying migratory properties (Fig. 7 C), control HeLa cells moved at a velocity of 14.1 ± 3.25 µm/h for a total distance of 125 ± 35.8 µm from their original position (n > 150). TTBK2 depletion decreased the velocity to 10.4 ± 3.48 µm/h and the total distance to 77.4 ± 37.4 µm (siTTBK2-1; n > 150). Essentially, similar results were obtained using a different TTBK2 siRNA (unpublished data). The expression of resTTBK2-WT rescued the inhibitory effects of TTBK2 depletion on HeLa cell migration (Fig. 7 C and Fig. S4 A), establishing the essential role of TTBK2 in these migratory properties. We then examined the effects of KIF2A depletion on cell migration and found that KIF2A depletion also inhibited the migration (Fig. 7, B and C; and Fig. S4 A). TTBK2 and KIF2A double depletion, however, partially rescued the migration defects caused by TTBK2 or KIF2A single depletion (Fig. 7, B and C; and Fig. S4 A). Considering that TTBK2 inactivates KIF2A, these results indicate that the proper activity level of KIF2A is required for cell migration, and activities of TTBK2 and KIF2A should be tightly balanced for effective cell migration.


TTBK2 with EB1/3 regulates microtubule dynamics in migrating cells through KIF2A phosphorylation.

Watanabe T, Kakeno M, Matsui T, Sugiyama I, Arimura N, Matsuzawa K, Shirahige A, Ishidate F, Nishioka T, Taya S, Hoshino M, Kaibuchi K - J. Cell Biol. (2015)

TTBK2 regulates cell migration via KIF2A. (A) Still images during wound healing of HeLa cells transfected with control or TTBK2 siRNA. TTBK2 depletion inhibited the migration of HeLa cells. (B) Migratory tracks of representative control, TTBK2-depleted, KIF2A-depleted, or TTBK2- and KIF2A-depleted cells. (C) Three parameters (migration distance, velocity, and directionality) were measured. For the control, TTBK2 depletion, and TTBK2/KIF2A double depletion, ≥150 cells from more than four independent experiments were analyzed. For KIF2A depletion or rescue with TTBK2-WT, ≥100 cells or ≥60 cells from three independent experiments were analyzed, respectively. ***, P < 0.001 (one-way ANOVA, Tukey’s HSD). (D) MT organization in migrating HeLa cells during wound healing. The boxes in the top panels are enlarged in the bottom panels. MT density at the cell front was measured as the mean intensity in the region indicated. TTBK2 depletion decreased MT intensity at the cell front. The decrease in MT intensity was reversed by co-depletion of KIF2A. Single-plane images focused on the cell periphery were used for the analysis. The fluorescence intensity was quantified in >20 cells for each condition and is shown as a ratio to the control cell value. The data represent the mean ± SD of three independent experiments (total of >60 cells for each condition). ***, P < 0.001 (one-way ANOVA, Tukey’s HSD). (E) The effects of TTBK2 depletion on the migration of cerebellar granule neurons in vivo. Granule neurons expressing control shRNA migrated toward the IGL through the ML, but neurons expressing TTBK2 shRNA exhibited only minimal migration. The graph shows the quantitative results. Error bars indicate the SEM. ***, P < 0.001 (one-way ANOVA). The data used for the statistical analysis were obtained from 12 slices of three brains (>300 cells). Bars: (A and B) 100 µm; (D) 10 µm; (D, magnification) 5 µm; (E) 50 µm.
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fig7: TTBK2 regulates cell migration via KIF2A. (A) Still images during wound healing of HeLa cells transfected with control or TTBK2 siRNA. TTBK2 depletion inhibited the migration of HeLa cells. (B) Migratory tracks of representative control, TTBK2-depleted, KIF2A-depleted, or TTBK2- and KIF2A-depleted cells. (C) Three parameters (migration distance, velocity, and directionality) were measured. For the control, TTBK2 depletion, and TTBK2/KIF2A double depletion, ≥150 cells from more than four independent experiments were analyzed. For KIF2A depletion or rescue with TTBK2-WT, ≥100 cells or ≥60 cells from three independent experiments were analyzed, respectively. ***, P < 0.001 (one-way ANOVA, Tukey’s HSD). (D) MT organization in migrating HeLa cells during wound healing. The boxes in the top panels are enlarged in the bottom panels. MT density at the cell front was measured as the mean intensity in the region indicated. TTBK2 depletion decreased MT intensity at the cell front. The decrease in MT intensity was reversed by co-depletion of KIF2A. Single-plane images focused on the cell periphery were used for the analysis. The fluorescence intensity was quantified in >20 cells for each condition and is shown as a ratio to the control cell value. The data represent the mean ± SD of three independent experiments (total of >60 cells for each condition). ***, P < 0.001 (one-way ANOVA, Tukey’s HSD). (E) The effects of TTBK2 depletion on the migration of cerebellar granule neurons in vivo. Granule neurons expressing control shRNA migrated toward the IGL through the ML, but neurons expressing TTBK2 shRNA exhibited only minimal migration. The graph shows the quantitative results. Error bars indicate the SEM. ***, P < 0.001 (one-way ANOVA). The data used for the statistical analysis were obtained from 12 slices of three brains (>300 cells). Bars: (A and B) 100 µm; (D) 10 µm; (D, magnification) 5 µm; (E) 50 µm.
Mentions: Finally, we examined whether TTBK2 regulates cell migration via KIF2A because appropriate MT dynamics and integrity are required for cell migration (Watanabe et al., 2005; Kuijpers and Hoogenraad, 2011; Etienne-Manneville, 2013). We performed wound healing assays using HeLa cells. The control HeLa cells migrated toward the gap during wound healing, but TTBK2 depletion inhibited this cell migration (Fig. 7 A). Tracking of each cell demonstrated the directional and persistent migration of control cells, whereas TTBK2-depleted cells migrated less efficiently, including a loss of directionality toward the wound (Fig. 7 B). When quantifying migratory properties (Fig. 7 C), control HeLa cells moved at a velocity of 14.1 ± 3.25 µm/h for a total distance of 125 ± 35.8 µm from their original position (n > 150). TTBK2 depletion decreased the velocity to 10.4 ± 3.48 µm/h and the total distance to 77.4 ± 37.4 µm (siTTBK2-1; n > 150). Essentially, similar results were obtained using a different TTBK2 siRNA (unpublished data). The expression of resTTBK2-WT rescued the inhibitory effects of TTBK2 depletion on HeLa cell migration (Fig. 7 C and Fig. S4 A), establishing the essential role of TTBK2 in these migratory properties. We then examined the effects of KIF2A depletion on cell migration and found that KIF2A depletion also inhibited the migration (Fig. 7, B and C; and Fig. S4 A). TTBK2 and KIF2A double depletion, however, partially rescued the migration defects caused by TTBK2 or KIF2A single depletion (Fig. 7, B and C; and Fig. S4 A). Considering that TTBK2 inactivates KIF2A, these results indicate that the proper activity level of KIF2A is required for cell migration, and activities of TTBK2 and KIF2A should be tightly balanced for effective cell migration.

Bottom Line: TTBK2 depletion reduced MT lifetime (facilitated shrinkage and suppressed rescue) and impaired HeLa cell migration, and these phenotypes were partially restored by KIF2A co-depletion.Expression of nonphosphorylatable KIF2A, but not wild-type KIF2A, reduced MT lifetime and slowed down the cell migration.These findings indicate that TTBK2 with EB1/3 phosphorylates KIF2A and antagonizes KIF2A-induced depolymerization at MT plus ends for cell migration.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cell Pharmacology, Nagoya University Graduate School of Medicine, Showa, Nagoya 466-8550, Japan.

Show MeSH
Related in: MedlinePlus