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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

Illustration of the signaling pathway involved in the effect of HYS-32 on microtubule catastrophes in rat astrocytes.HYS-32 induces microtubule catastrophes by causing EB1 dislodgement from microtubule plus ends and EB1 accumulation on the microtubule lattice through the modulation of the PI3K-GSK3β signaling pathway.
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pone.0126217.g011: Illustration of the signaling pathway involved in the effect of HYS-32 on microtubule catastrophes in rat astrocytes.HYS-32 induces microtubule catastrophes by causing EB1 dislodgement from microtubule plus ends and EB1 accumulation on the microtubule lattice through the modulation of the PI3K-GSK3β signaling pathway.

Mentions: In conclusion, we have demonstrated that a HYS-32-induced microtubule catastrophe causes EB1 dislodgement from microtubule plus ends and EB1 accumulation on the microtubule lattice through the modulation of the PI3K-GSK3β signaling pathway (Fig 11). The novel biological efficacy of HYS-32 on microtubule dynamic instability in astrocytes may present as a new potential therapeutic drug for axonal regeneration around the glial scar following severe CNS injuries.


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)

Illustration of the signaling pathway involved in the effect of HYS-32 on microtubule catastrophes in rat astrocytes.HYS-32 induces microtubule catastrophes by causing EB1 dislodgement from microtubule plus ends and EB1 accumulation on the microtubule lattice through the modulation of the PI3K-GSK3β signaling pathway.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4418738&req=5

pone.0126217.g011: Illustration of the signaling pathway involved in the effect of HYS-32 on microtubule catastrophes in rat astrocytes.HYS-32 induces microtubule catastrophes by causing EB1 dislodgement from microtubule plus ends and EB1 accumulation on the microtubule lattice through the modulation of the PI3K-GSK3β signaling pathway.
Mentions: In conclusion, we have demonstrated that a HYS-32-induced microtubule catastrophe causes EB1 dislodgement from microtubule plus ends and EB1 accumulation on the microtubule lattice through the modulation of the PI3K-GSK3β signaling pathway (Fig 11). The novel biological efficacy of HYS-32 on microtubule dynamic instability in astrocytes may present as a new potential therapeutic drug for axonal regeneration around the glial scar following severe CNS injuries.

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