MZT1 regulates microtubule nucleation by linking γTuRC assembly to adapter-mediated targeting and activation.

Regulation of the γ-tubulin ring complex (γTuRC) through targeting and activation restricts nucleation of microtubules to microtubule-organizing centers (MTOCs), aiding in the assembly of ordered microtubule arrays. However, the mechanistic basis of this important regulation remains poorly understood. Here, we show that, in human cells, γTuRC integrity, determined by the presence of γ-tubulin complex proteins (GCPs; also known as TUBGCPs) 2-6, is a prerequisite for interaction with the targeting factor NEDD1, impacting on essentially all γ-tubulin-dependent functions. Recognition of γTuRC integrity is mediated by MZT1, which binds not only to the GCP3 subunit as previously shown, but cooperatively also to other GCPs through a conserved hydrophobic motif present in the N-termini of GCP2, GCP3, GCP5 and GCP6. MZT1 knockdown causes severe cellular defects under conditions that leave γTuRC intact, suggesting that the essential function of MZT1 is not in γTuRC assembly. Instead, MZT1 specifically binds fully assembled γTuRC to enable interaction with NEDD1 for targeting, and with the CM1 domain of CDK5RAP2 for stimulating nucleation activity. Thus, MZT1 is a 'priming factor' for γTuRC that allows spatial regulation of nucleation.

The where, when and how of microtubule nucleation - one ring to rule them all.

The function of microtubules depends on their arrangement into highly ordered arrays. Spatio-temporal control over the formation of new microtubules and regulation of their properties are central to the organization of these arrays. The nucleation of new microtubules requires γ-tubulin, an essential protein that assembles into multi-subunit complexes and is found in all eukaryotic organisms. However, the way in which γ-tubulin complexes are regulated and how this affects nucleation and, potentially, microtubule behavior, is poorly understood. γ-tubulin has been found in complexes of various sizes but several lines of evidence suggest that only large, ring-shaped complexes function as efficient microtubule nucleators. Human γ-tubulin ring complexes (γTuRCs) are composed of γ-tubulin and the γ-tubulin complex components (GCPs) 2, 3, 4, 5 and 6, which are members of a conserved protein family. Recent work has identified additional unrelated γTuRC subunits, as well as a large number of more transient γTuRC interactors. In this Commentary, we discuss the regulation of γTuRC-dependent microtubule nucleation as a key mechanism of microtubule organization. Specifically, we focus on the regulatory roles of the γTuRC subunits and interactors and present an overview of other mechanisms that regulate γTuRC-dependent microtubule nucleation and organization.

SPICE--a previously uncharacterized protein required for centriole duplication and mitotic chromosome congression.

Proper assembly and function of a bipolar mitotic spindle is crucial for faithful bidirectional chromosome segregation during cell division. In animal cells, the two poles of the mitotic spindle are organized by centrosomes, microtubule-organizing structures composed of a pair of centrioles surrounded by the so-called pericentriolar material. Proteomic studies have revealed a large number of centrosome proteins, but many remain uncharacterized. Here, we characterize SPICE, a protein that localizes to spindle microtubules in mitosis and to centrioles throughout the cell cycle. RNAi-mediated depletion of SPICE in human cells impairs centriole duplication and causes severe mitotic defects. SPICE depletion compromises spindle architecture, spindle pole integrity and chromosome congression, even in cells in which centriole duplication has occurred. Our data suggest that SPICE is an important dual-function regulator required for centriole duplication and for proper bipolar spindle formation and chromosome congression in mitosis.

The gammaTuRC revisited: a comparative analysis of interphase and mitotic human gammaTuRC redefines the set of core components and identifies the novel subunit GCP8.

The γ-tubulin complex is a multi-subunit protein complex that nucleates microtubule polymerization. γ-Tubulin complexes are present in all eukaryotes, but size and subunit composition vary. In Drosophila, Xenopus, and humans large γ-tubulin ring complexes (γTuRCs) have been described, which have a characteristic open ring-shaped structure and are composed of a similar set of subunits, named γ-tubulin, GCPs 2-6, and GCP-WD in humans. Despite the identification of these proteins, γTuRC function and regulation remain poorly understood. Here we establish a new method for the purification of native human γTuRC. Using mass spectrometry of whole protein mixtures we compared the composition of γTuRCs from nonsynchronized and mitotic human cells. Based on our analysis we can define core subunits as well as more transient interactors such as the augmin complex, which associates specifically with mitotic γTuRCs. We also identified GCP8/MOZART2 as a novel core subunit that is present in both interphase and mitotic γTuRCs. GCP8 depletion does not affect γTuRC assembly but interferes with γTuRC recruitment and microtubule nucleation at interphase centrosomes without disrupting general centrosome structure. GCP8-depleted cells do not display any obvious mitotic defects, suggesting that GCP8 specifically affects the organization of the interphase microtubule network.

Small carboxyl-terminal domain phosphatase 2 attenuates androgen-dependent transcription.

Small carboxyl-terminal domain (CTD) phosphatase 2 (SCP2) was identified and verified as a protein that interacts with the androgen receptor (AR). Ectopic expression of SCP2 or two other family members, SCP1 and SCP3, attenuated AR transcriptional activity in LNCaP cells and were recruited in an androgen- and AR-dependent fashion onto the prostate-specific antigen (PSA) promoter. Silencing SCP2 and SCP1 by short hairpin RNAs increased androgen-dependent transcription of the PSA gene and augmented AR loading onto the PSA promoter and enhancer. SCP2 also attenuated glucocorticoid receptor (GR) function, and its silencing increased dexamethasone-mediated PSA mRNA accumulation and GR loading onto the PSA enhancer in LNCaP 1F5 cells. SCP2 silencing was accompanied by augmented recruitment and earlier cycling of RNA polymerase II on the promoter. Ser 5 phosphorylation of the RNA polymerase II CTD, a process necessary for initiation of transcription elongation, occurred significantly earlier in SCP2-silenced than parental LNCaP cells. Collectively, our results suggest that SCP2 is involved in promoter clearance during steroid-activated transcription.