PLoS ONE (Jan 2015)

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

  • Chi-Ting Chiu,
  • Chih-Kai Liao,
  • Chien-Chang Shen,
  • Tswen-Kei Tang,
  • Guey-Mei Jow,
  • Hwai-Shi Wang,
  • Jiahn-Chun Wu

DOI
https://doi.org/10.1371/journal.pone.0126217
Journal volume & issue
Vol. 10, no. 5
p. e0126217

Abstract

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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.