Abstract
Pregnenolone (P5) is a neurosteroid that improves memory and neurological recovery. It is also required for zebrafish embryonic development. However, its mode of action is unclear. Here we show that P5 promotes cell migration and microtubule polymerization by binding a microtubule plus end–tracking protein, cytoplasmic linker protein 1 (CLIP-170). We captured CLIP-170 from zebrafish embryonic extract using a P5 photoaffinity probe conjugated to diaminobenzophenone. P5 interacted with CLIP-170 at its coiled-coil domain and changed it into an extended conformation. This increased CLIP-170 interaction with microtubules, dynactin subunit p150Glued and LIS1; it also promoted CLIP-170–dependent microtubule polymerization. CLIP-170 was essential for P5 to promote microtubule abundance and zebrafish epiboly cell migration during embryogenesis, and overexpression of the P5-binding region of CLIP-170 delayed this migration. P5 also sustained migration directionality of cultured mammalian cells. Our results show that P5 activates CLIP-170 to promote microtubule polymerization and cell migration.
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Acknowledgements
We thank S. Takashima (Osaka University) and N. Galjart (Erasmus Medical Center) for CLIP-170 plasmids, S. Tsukita (Kyoto University) for the EB1-GFP plasmid, S. Halpain (University of California–San Diego) for the MAP2c plasmid and Y. Mimori-Kiyosue (RIKEN) for the GFP-CLIP-115 plasmid, generated by A. Akhmanova (Utrecht University). We thank W.-y. Ku and C.-w. Hsu for help in drawing the graphical abstract. RNAi reagents were obtained from the National RNAi Core Facility Platform at Academia Sinica (NSC 97-3112-B-001-016). The wild-type zebrafish were from Taiwan Zebrafish Core Facility at Academia Sinica (NSC 100-2321-B-001-030). C.-T.C. was supported by the National Science Council (NSC 97-2113-M-002-002-MY3 and NSC99-3112-B-001-002) and the National Taiwan University, and B.-c.C. was supported by the National Science Council (NSC101-2321-B-001-001, NSC 102-2311-B-001-013-MY3), the National Health Research Institute (NHRI-EX102-10210SI) and Academia Sinica.
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J.-H.W. designed the study, performed most of the experiments and wrote the manuscript; M.-R.L. synthesized P5s-NBPN, OH-NBPN and Cho-NBPN; C.-H.C. synthesized P5-NBPN and 7-OH-P5; S.-K.T. examined zebrafish clip1 expression patterns; T.-C.H. synthesized P5β-NBPN; S.-P.L. stained CLIP-170 for EM analysis; Y.-R.C. performed LC/MS/MS identification; C.-T.C. devised P5-photoaffinity probes and supervised syntheses of P5 derivatives; B.-c.C. designed the study, oversaw its execution and wrote the manuscript.
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Weng, JH., Liang, MR., Chen, CH. et al. Pregnenolone activates CLIP-170 to promote microtubule growth and cell migration. Nat Chem Biol 9, 636–642 (2013). https://doi.org/10.1038/nchembio.1321
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DOI: https://doi.org/10.1038/nchembio.1321
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