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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

LY294002 inhibits the HYS-32-induced phosphorylation of GSK3β-pS9 and GSK3β-pY216.(A) Control astrocytes (Con) or astrocytes treated for 24 h with 5 μM HYS-32 (HYS), co-treated for 24 h with 5 μM HYS-32 and 20 μM LY294002 (HYS+LY), or treated with 20 μM LY294002 (LY) were subjected to 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against GSK3β-pSer9, GSK3β-pY216, totalGSK3β, or GAPDH. (B) Densitometric analyses of GSK3β-pSer9 and GSK3β-pY216 expressed as the density of the bands in the treated groups relative to the control. (C) According to the data in (B), the stacked bar graph showing relative percentage ofphospho-GSK3β in various treatment. (D)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-hoctest.
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pone.0126217.g009: LY294002 inhibits the HYS-32-induced phosphorylation of GSK3β-pS9 and GSK3β-pY216.(A) Control astrocytes (Con) or astrocytes treated for 24 h with 5 μM HYS-32 (HYS), co-treated for 24 h with 5 μM HYS-32 and 20 μM LY294002 (HYS+LY), or treated with 20 μM LY294002 (LY) were subjected to 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against GSK3β-pSer9, GSK3β-pY216, totalGSK3β, or GAPDH. (B) Densitometric analyses of GSK3β-pSer9 and GSK3β-pY216 expressed as the density of the bands in the treated groups relative to the control. (C) According to the data in (B), the stacked bar graph showing relative percentage ofphospho-GSK3β in various treatment. (D)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-hoctest.

Mentions: We next examined whether the effects of HYS-32 on EB1 distribution in astrocytes were mediated through the GSK3β-pS9 signaling pathway by inhibiting PI3K. As shown in Fig 9, treatment of astrocytes with HYS-32 for 24 h (Fig 9A and 9B, HYS) caused a significant increase in both GSK3β-pS9 and GSK3β-pY216 protein levels as compared to the control astrocytes (Fig 9A and 9B, Con). The HYS-32-induced elevation in GSK3β-pS9 and GSK3β-pY216 levels was both inhibited by co-treatment with PI3K inhibitor LY294002 in HYS-32-treated astrocytes (Fig 9A and 9B, HYS+LY). Ratio of GSK3β-pY216 to GSK3β-pS9 (Y216/S9) was about 0.97 in control astrocytes (Fig 9C, Con), declined to 0.75 after 24 h of HYS-32 treatments (Fig 9C, HYS), and back to 0.91 by co-treatment with HYS-32 and LY294002 (Fig 9C, HYS+LY). Levels of total GSK3β protein were unchanged in LY294002-treated astrocytes as demonstrated by immunoblot analysis (Fig 9A). HYS-32 treatment caused a significant increase in distance between microtubule tips and cell border (Fig 9D, HYS) as compared to the controls (Fig 9D, Con). The microtubule tips and the cell border distances increased by HYS-32 were significantly decreased by co-treatment with LY294002 in the HYS-32-treated astrocytes (Fig 9D, HYS+LY). Double immunofluorescence confocal microscopy showed that the HYS-32-induced depletion of EB1 at microtubule plus end was partially recovered by co-treatment of LY294002 in HYS-32-treatedastrocytes (Fig 8, HYS+LY; compare arrowheads and double arrowheads in Enlarged). Treatment of LY294002 alone had little effect on the distance between microtubule tips and the cell border (Fig 9D, LY) and EB1 distribution (Fig 8, LY) 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)

LY294002 inhibits the HYS-32-induced phosphorylation of GSK3β-pS9 and GSK3β-pY216.(A) Control astrocytes (Con) or astrocytes treated for 24 h with 5 μM HYS-32 (HYS), co-treated for 24 h with 5 μM HYS-32 and 20 μM LY294002 (HYS+LY), or treated with 20 μM LY294002 (LY) were subjected to 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against GSK3β-pSer9, GSK3β-pY216, totalGSK3β, or GAPDH. (B) Densitometric analyses of GSK3β-pSer9 and GSK3β-pY216 expressed as the density of the bands in the treated groups relative to the control. (C) According to the data in (B), the stacked bar graph showing relative percentage ofphospho-GSK3β in various treatment. (D)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-hoctest.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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pone.0126217.g009: LY294002 inhibits the HYS-32-induced phosphorylation of GSK3β-pS9 and GSK3β-pY216.(A) Control astrocytes (Con) or astrocytes treated for 24 h with 5 μM HYS-32 (HYS), co-treated for 24 h with 5 μM HYS-32 and 20 μM LY294002 (HYS+LY), or treated with 20 μM LY294002 (LY) were subjected to 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against GSK3β-pSer9, GSK3β-pY216, totalGSK3β, or GAPDH. (B) Densitometric analyses of GSK3β-pSer9 and GSK3β-pY216 expressed as the density of the bands in the treated groups relative to the control. (C) According to the data in (B), the stacked bar graph showing relative percentage ofphospho-GSK3β in various treatment. (D)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-hoctest.
Mentions: We next examined whether the effects of HYS-32 on EB1 distribution in astrocytes were mediated through the GSK3β-pS9 signaling pathway by inhibiting PI3K. As shown in Fig 9, treatment of astrocytes with HYS-32 for 24 h (Fig 9A and 9B, HYS) caused a significant increase in both GSK3β-pS9 and GSK3β-pY216 protein levels as compared to the control astrocytes (Fig 9A and 9B, Con). The HYS-32-induced elevation in GSK3β-pS9 and GSK3β-pY216 levels was both inhibited by co-treatment with PI3K inhibitor LY294002 in HYS-32-treated astrocytes (Fig 9A and 9B, HYS+LY). Ratio of GSK3β-pY216 to GSK3β-pS9 (Y216/S9) was about 0.97 in control astrocytes (Fig 9C, Con), declined to 0.75 after 24 h of HYS-32 treatments (Fig 9C, HYS), and back to 0.91 by co-treatment with HYS-32 and LY294002 (Fig 9C, HYS+LY). Levels of total GSK3β protein were unchanged in LY294002-treated astrocytes as demonstrated by immunoblot analysis (Fig 9A). HYS-32 treatment caused a significant increase in distance between microtubule tips and cell border (Fig 9D, HYS) as compared to the controls (Fig 9D, Con). The microtubule tips and the cell border distances increased by HYS-32 were significantly decreased by co-treatment with LY294002 in the HYS-32-treated astrocytes (Fig 9D, HYS+LY). Double immunofluorescence confocal microscopy showed that the HYS-32-induced depletion of EB1 at microtubule plus end was partially recovered by co-treatment of LY294002 in HYS-32-treatedastrocytes (Fig 8, HYS+LY; compare arrowheads and double arrowheads in Enlarged). Treatment of LY294002 alone had little effect on the distance between microtubule tips and the cell border (Fig 9D, LY) and EB1 distribution (Fig 8, LY) 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