Abstract
BackgroundAstral microtubules emanating from the mitotic centrosomes play pivotal roles in defining cell division axis and tissue morphogenesis. Previous studies have demonstrated that human transforming acidic coiled-coil 3 (TACC3), the most conserved TACC family protein, regulates formation of astral microtubules at centrosomes in vertebrate cells by affecting γ-tubulin ring complex (γ-TuRC) assembly. However, the molecular mechanisms underlying such function were not completely understood.ResultsHere, we show that Aurora A site-specific phosphorylation in TACC3 regulates formation of astral microtubules by stabilizing γ-TuRC assembly in human cells. Mutation of the most conserved Aurora A targeting site, Ser 558 to alanine (S558A) in TACC3 results in robust loss of astral microtubules and disrupts localization of the γ-tubulin ring complex (γ-TuRC) proteins at the spindle poles. Under similar condition, phospho-mimicking S558D mutation retains astral microtubules and the γ-TuRC proteins in a manner similar to control cells expressed with wild type TACC3. Time-lapse imaging reveals that S558A mutation leads to defects in positioning of the spindle-poles and thereby causes delay in metaphase to anaphase transition. Biochemical results determine that the Ser 558- phosphorylated TACC3 interacts with the γ-TuRC proteins and further, S558A mutation impairs the interaction. We further reveal that the mutation affects the assembly of γ-TuRC from the small complex components.ConclusionsThe results demonstrate that TACC3 phosphorylation stabilizes γ- tubulin ring complex assembly and thereby regulates formation of centrosomal asters. They also implicate a potential role of TACC3 phosphorylation in the functional integrity of centrosomes/spindle poles.
Highlights
Astral microtubules emanating from the mitotic centrosomes play pivotal roles in defining cell division axis and tissue morphogenesis
We have investigated the role of transforming acidic coiledcoil 3 (TACC3) phosphorylation in regulation of the γ-tubulin ring complexes and sought to determine its molecular link with astral microtubule assembly at the centrosomes in human cells
Phosphorylated TACC3 is required for formation of astral microtubules at the centrosomes/spindle poles We first determined whether phosphorylation at Ser 558 site of human TACC3 imparts any role in the TACC3mediated microtubule assembly at the centrosomes in mitotic cells
Summary
Astral microtubules emanating from the mitotic centrosomes play pivotal roles in defining cell division axis and tissue morphogenesis. G2/M transition and the process involves recruitment of many structural and regulatory proteins to the PCM [1]. Critical among those are γ-tubulin and its associated protein complexes that are essential for microtubule nucleation from the centrosomes. Γ-tubulin and conserved γ-tubulin-binding proteins, GCP2 and GCP3 form a sub-complex called γ-tubulin-small complex (γ-TuSC) Multiple such γ-TuSCs together with additional GCP proteins, such as GCP 4, 5 and 6, assemble to constitute the larger γ-tubulin ring complex (γ-TuRC), which serves as the main microtubule-nucleating machinery in cells [2,3,4]. Centrosomal recruitment of γ-tubulin increases ~ 3–5-folds as cells enter M phase [5] and such
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