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HYS-32-Induced Microtubule Catastrophes in Rat Astrocytes Involves the PI3K-GSK3beta Signaling Pathway.

Chiu CT, Liao CK, Shen CC, Tang TK, Jow GM, Wang HS, Wu JC - PLoS ONE (2015)

Bottom Line: Time-lapse experiments with immunoprecipitation further displayed that the association between EB-1 and β-tubulin was significantly decreased following a short-term treatment (2 h), but gradually increased in a prolonged treatment (6-24 h) with HYS-32.Further, HYS-32 treatment induced GSK3β phosphorylation at Y216 and S9, where the ratio of GSK3β-pY216 to GSK3β-pS9 was first elevated followed by a decrease over time.Together these findings suggest that HYS-32 induces microtubule catastrophes by preventing EB1 from targeting to microtubule plus ends through the GSK3β signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Institute of Anatomy and Cell Biology, School of Medicine, National Yang-Ming University, Taipei 11221, Taiwan.

ABSTRACT
HYS-32 is a novel derivative of combretastatin-A4 (CA-4) previously shown to induce microtubule coiling in rat primary astrocytes. In this study, we further investigated the signaling mechanism and EB1, a microtubule-associated end binding protein, involved in HYS-32-induced microtubule catastrophes. Confocal microscopy with double immunofluorescence staining revealed that EB1 accumulates at the growing microtubule plus ends, where they exhibit a bright comet-like staining pattern in control astrocytes. HYS-32 induced microtubule catastrophes in both a dose- and time-dependent manner and dramatically increased the distances between microtubule tips and the cell border. Treatment of HYS-32 (5 μM) eliminated EB1 localization at the microtubule plus ends and resulted in an extensive redistribution of EB1 to the microtubule lattice without affecting the β-tubulin or EB1 protein expression. Time-lapse experiments with immunoprecipitation further displayed that the association between EB-1 and β-tubulin was significantly decreased following a short-term treatment (2 h), but gradually increased in a prolonged treatment (6-24 h) with HYS-32. Further, HYS-32 treatment induced GSK3β phosphorylation at Y216 and S9, where the ratio of GSK3β-pY216 to GSK3β-pS9 was first elevated followed by a decrease over time. Co-treatment of astrocytes with HYS-32 and GSK3β inhibitor SB415286 attenuated the HYS-32-induced microtubule catastrophes and partially prevented EB1 dissociation from the plus end of microtubules. Furthermore, co-treatment with PI3K inhibitor LY294002 inhibited HYS-32-induced GSK3β-pS9 and partially restored EB1 distribution from the microtubule lattice to plus ends. Together these findings suggest that HYS-32 induces microtubule catastrophes by preventing EB1 from targeting to microtubule plus ends through the GSK3β signaling pathway.

No MeSH data available.


Related in: MedlinePlus

GSK3β inhibitor SB415286 inhibits the HYS-32-induced phosphorylation of GSK3β-pY216 and attenuates the HYS-32-induced microtubule catastrophes.(A) Control astrocytes (Con) or astrocytes treated for 24 h with 5 μM HYS-32 (HYS), co-treated for 24 h with 5μM HYS-32and 20μM SB415286 (HYS+SB), or treated with 20 μM SB415286 (SB) were subjected to 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against GSK3β-pTyr216 or GAPDH. (B) Densitometric analyses of GSK3β-pTyr216 expressed as the density of the bands in the treated groups relative to the control. (C) Astrocytes treated as in (A) were fixed in cold acetone and triple-stained for β-tubulin, N-cadherin, and F-actin. Quantitative analysis of the straight distance between microtubule tips and cell border were performed as described in Materials and Methods. The results were collected from three independent experiments. *p<0.01 compared to HYS using one-way ANOVA with Dunnett’s post-hoc test.
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pone.0126217.g007: GSK3β inhibitor SB415286 inhibits the HYS-32-induced phosphorylation of GSK3β-pY216 and attenuates the HYS-32-induced microtubule catastrophes.(A) Control astrocytes (Con) or astrocytes treated for 24 h with 5 μM HYS-32 (HYS), co-treated for 24 h with 5μM HYS-32and 20μM SB415286 (HYS+SB), or treated with 20 μM SB415286 (SB) were subjected to 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against GSK3β-pTyr216 or GAPDH. (B) Densitometric analyses of GSK3β-pTyr216 expressed as the density of the bands in the treated groups relative to the control. (C) Astrocytes treated as in (A) were fixed in cold acetone and triple-stained for β-tubulin, N-cadherin, and F-actin. Quantitative analysis of the straight distance between microtubule tips and cell border were performed as described in Materials and Methods. The results were collected from three independent experiments. *p<0.01 compared to HYS using one-way ANOVA with Dunnett’s post-hoc test.

Mentions: We next examined whether the effects of HYS-32 on microtubule catastrophes and EB1 distribution in astrocytes were mediated through the GSK3β-pY216 signaling pathway. As shown by the immunoblot analysis in Fig 7, treatment of astrocytes with HYS-32 for 24 h (Fig 7A and 7B, HYS) caused a notable increase in GSK3β-pY216 levels when compared to control astrocytes (Fig 7A and 7B, Con). The HYS-32-induced increase in GSK3β-pY216 protein levels were inhibited by co-treatment with GSK3β inhibitor SB415286 in HYS-32-treated astrocytes (Fig 7A and 7B, HYS+SB). Quantitative analyses showed that HYS-32 treatment caused a significant increase in distance between microtubule tips and the cell border (Fig 7C, HYS) as compared to the controls (Fig 7C, Con). The HYS-32-increased microtubule tips and cell border distances were greatly decreased by co-treatment with SB415286 in the HYS-32-treated astrocytes (Fig 7C, HYS+SB); however, confocal microscopy revealed that the HYS-32-induced depletion of EB1 comet-like streaks (Fig 8, HYS, arrowheads) on microtubule plus ends were only partially recovered by co-treatment of SB415286 in HYS-32-treated astrocytes (Fig 8, HYS+SB; compare arrowheads and double arrowheads in Enlarged). Treatment of SB415286 alone had no effects on microtubule tips and the cell border distance (Fig 7C, SB) and EB1 distribution (Fig 8, SB) in astrocytes.


HYS-32-Induced Microtubule Catastrophes in Rat Astrocytes Involves the PI3K-GSK3beta Signaling Pathway.

Chiu CT, Liao CK, Shen CC, Tang TK, Jow GM, Wang HS, Wu JC - PLoS ONE (2015)

GSK3β inhibitor SB415286 inhibits the HYS-32-induced phosphorylation of GSK3β-pY216 and attenuates the HYS-32-induced microtubule catastrophes.(A) Control astrocytes (Con) or astrocytes treated for 24 h with 5 μM HYS-32 (HYS), co-treated for 24 h with 5μM HYS-32and 20μM SB415286 (HYS+SB), or treated with 20 μM SB415286 (SB) were subjected to 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against GSK3β-pTyr216 or GAPDH. (B) Densitometric analyses of GSK3β-pTyr216 expressed as the density of the bands in the treated groups relative to the control. (C) Astrocytes treated as in (A) were fixed in cold acetone and triple-stained for β-tubulin, N-cadherin, and F-actin. Quantitative analysis of the straight distance between microtubule tips and cell border were performed as described in Materials and Methods. The results were collected from three independent experiments. *p<0.01 compared to HYS using one-way ANOVA with Dunnett’s post-hoc test.
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pone.0126217.g007: GSK3β inhibitor SB415286 inhibits the HYS-32-induced phosphorylation of GSK3β-pY216 and attenuates the HYS-32-induced microtubule catastrophes.(A) Control astrocytes (Con) or astrocytes treated for 24 h with 5 μM HYS-32 (HYS), co-treated for 24 h with 5μM HYS-32and 20μM SB415286 (HYS+SB), or treated with 20 μM SB415286 (SB) were subjected to 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against GSK3β-pTyr216 or GAPDH. (B) Densitometric analyses of GSK3β-pTyr216 expressed as the density of the bands in the treated groups relative to the control. (C) Astrocytes treated as in (A) were fixed in cold acetone and triple-stained for β-tubulin, N-cadherin, and F-actin. Quantitative analysis of the straight distance between microtubule tips and cell border were performed as described in Materials and Methods. The results were collected from three independent experiments. *p<0.01 compared to HYS using one-way ANOVA with Dunnett’s post-hoc test.
Mentions: We next examined whether the effects of HYS-32 on microtubule catastrophes and EB1 distribution in astrocytes were mediated through the GSK3β-pY216 signaling pathway. As shown by the immunoblot analysis in Fig 7, treatment of astrocytes with HYS-32 for 24 h (Fig 7A and 7B, HYS) caused a notable increase in GSK3β-pY216 levels when compared to control astrocytes (Fig 7A and 7B, Con). The HYS-32-induced increase in GSK3β-pY216 protein levels were inhibited by co-treatment with GSK3β inhibitor SB415286 in HYS-32-treated astrocytes (Fig 7A and 7B, HYS+SB). Quantitative analyses showed that HYS-32 treatment caused a significant increase in distance between microtubule tips and the cell border (Fig 7C, HYS) as compared to the controls (Fig 7C, Con). The HYS-32-increased microtubule tips and cell border distances were greatly decreased by co-treatment with SB415286 in the HYS-32-treated astrocytes (Fig 7C, HYS+SB); however, confocal microscopy revealed that the HYS-32-induced depletion of EB1 comet-like streaks (Fig 8, HYS, arrowheads) on microtubule plus ends were only partially recovered by co-treatment of SB415286 in HYS-32-treated astrocytes (Fig 8, HYS+SB; compare arrowheads and double arrowheads in Enlarged). Treatment of SB415286 alone had no effects on microtubule tips and the cell border distance (Fig 7C, SB) and EB1 distribution (Fig 8, SB) in astrocytes.

Bottom Line: Time-lapse experiments with immunoprecipitation further displayed that the association between EB-1 and β-tubulin was significantly decreased following a short-term treatment (2 h), but gradually increased in a prolonged treatment (6-24 h) with HYS-32.Further, HYS-32 treatment induced GSK3β phosphorylation at Y216 and S9, where the ratio of GSK3β-pY216 to GSK3β-pS9 was first elevated followed by a decrease over time.Together these findings suggest that HYS-32 induces microtubule catastrophes by preventing EB1 from targeting to microtubule plus ends through the GSK3β signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Institute of Anatomy and Cell Biology, School of Medicine, National Yang-Ming University, Taipei 11221, Taiwan.

ABSTRACT
HYS-32 is a novel derivative of combretastatin-A4 (CA-4) previously shown to induce microtubule coiling in rat primary astrocytes. In this study, we further investigated the signaling mechanism and EB1, a microtubule-associated end binding protein, involved in HYS-32-induced microtubule catastrophes. Confocal microscopy with double immunofluorescence staining revealed that EB1 accumulates at the growing microtubule plus ends, where they exhibit a bright comet-like staining pattern in control astrocytes. HYS-32 induced microtubule catastrophes in both a dose- and time-dependent manner and dramatically increased the distances between microtubule tips and the cell border. Treatment of HYS-32 (5 μM) eliminated EB1 localization at the microtubule plus ends and resulted in an extensive redistribution of EB1 to the microtubule lattice without affecting the β-tubulin or EB1 protein expression. Time-lapse experiments with immunoprecipitation further displayed that the association between EB-1 and β-tubulin was significantly decreased following a short-term treatment (2 h), but gradually increased in a prolonged treatment (6-24 h) with HYS-32. Further, HYS-32 treatment induced GSK3β phosphorylation at Y216 and S9, where the ratio of GSK3β-pY216 to GSK3β-pS9 was first elevated followed by a decrease over time. Co-treatment of astrocytes with HYS-32 and GSK3β inhibitor SB415286 attenuated the HYS-32-induced microtubule catastrophes and partially prevented EB1 dissociation from the plus end of microtubules. Furthermore, co-treatment with PI3K inhibitor LY294002 inhibited HYS-32-induced GSK3β-pS9 and partially restored EB1 distribution from the microtubule lattice to plus ends. Together these findings suggest that HYS-32 induces microtubule catastrophes by preventing EB1 from targeting to microtubule plus ends through the GSK3β signaling pathway.

No MeSH data available.


Related in: MedlinePlus