Abstract
Inhibitors of microtubule (MT) assembly or dynamics that target α/β-tubulin are widely exploited in cancer therapy and biological research. However, specific inhibitors of the MT nucleator γ-tubulin that would allow testing temporal functions of γ-tubulin during the cell cycle are yet to be identified. By evolving β-tubulin-binding drugs we now find that the glaziovianin A derivative gatastatin is a γ-tubulin-specific inhibitor. Gatastatin decreased interphase MT dynamics of human cells without affecting MT number. Gatastatin inhibited assembly of the mitotic spindle in prometaphase. Addition of gatastatin to preformed metaphase spindles altered MT dynamics, reduced the number of growing MTs and shortened spindle length. Furthermore, gatastatin prolonged anaphase duration by affecting anaphase spindle structure, indicating the continuous requirement of MT nucleation during mitosis. Thus, gatastatin facilitates the dissection of the role of γ-tubulin during the cell cycle and reveals the sustained role of γ-tubulin.
Highlights
Inhibitors of microtubule (MT) assembly or dynamics that target a/b-tubulin are widely exploited in cancer therapy and biological research
While a/b-tubulin heterodimers can spontaneously polymerize to generate MTs in vitro, MT nucleation in vivo is initiated from a ring-like template of g-tubulin that can promote MT nucleation at concentrations below those required for spontaneous assembly1–3. g-Tubulin recruits accessory proteins, so-called g-tubulin complex proteins (GCPs). g-Tubulin, GCP2 and GCP3 form a tetrameric 2:1:1 complex named the small g-tubulin complex (g-TuSC)
We lack a clear understanding of the requirements of g-tubulin at discrete cell cycle phases that arises from acute inhibition of g-tubulin functions through pharmacological intervention
Summary
Inhibitors of microtubule (MT) assembly or dynamics that target a/b-tubulin are widely exploited in cancer therapy and biological research. We showed that at an AG1 concentration that inhibits MT polymerization (Fig. 1b,c and Supplementary Fig. 3a–c), it failed to affect GTP-binding to g-tubulin (Table 2). The ratio of the halfmaximal inhibitory concentration (IC50) values confirmed that gatastatin inhibits g-tubulin-induced aster formation while AG1 is a general tubulin polymerization inhibitor (Fig. 1f; IC50[DMSO-aster]/IC50[RanQ69L-aster] 1⁄4 410.4 and 3.14, respectively).
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