HYS-32-Induced Microtubule Catastrophes in Rat Astrocytes Involves the PI3K-GSK3beta Signaling Pathway.
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.
Institute of Anatomy and Cell Biology, School of Medicine, National Yang-Ming University, Taipei 11221, Taiwan.
Chi-Ting
CT
Chiu
Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Taipei 10051, Taiwan.
Chih-Kai
CK
Liao
Division of Medicinal Chemistry, National Research Institute of Chinese Medicine, Taipei 11221, Taiwan.
Chien-Chang
CC
Shen
Department of Nursing, College of Health and Nursing, National Quemoy University, Kinmen 89250, Taiwan.
Tswen-Kei
TK
Tang
School of Medicine, Fu-Jen Catholic University, New Taipei City 24205, Taiwan.
Guey-Mei
GM
Jow
Institute of Anatomy and Cell Biology, School of Medicine, National Yang-Ming University, Taipei 11221, Taiwan.
Hwai-Shi
HS
Wang
Institute of Anatomy and Cell Biology, School of Medicine, National Yang-Ming University, Taipei 11221, Taiwan.
Jiahn-Chun
JC
Wu
D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000494:pharmacology; D000627:Aminophenols / Q000494:pharmacology; D000818:Animals; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism*; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D008301:Maleimides / Q000494:pharmacology; D008870:Microtubules / Q000378:metabolism*; D009281:Naphthalenes / Q000494:pharmacology*; D018696:Neuroprotective Agents / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects*
D015107:4-Butyrolactone; D000627:Aminophenols; D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D008301:Maleimides; D009281:Naphthalenes; D018696:Neuroprotective Agents; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; C417520:3-(3-chloro-4-hydroxyphenylamino)-4-(4-nitrophenyl)-1H-pyrrole-2,5-dione; C583851:4-(3,4-dimethoxyphenyl)-3-(naphthalen-2-yl)-2(5H)-furanone; C000605508:Gsk3b protein, rat