The γTuRC Revisited: A Comparative Analysis of Interphase and Mitotic Human γTuRC Redefines the Set of Core Components and Identifies the Novel Subunit GCP8

We compared the composition of γ-tubulin ring complexes tandem-affinity purified from asynchronous and mitotic human cells by mass spectrometry. We identified various interactors including the novel core subunit GCP8. GCP8 is the first subunit with an interphase-specific role in centrosomal γ-tubulin recruitment and microtubule nucleation

Noncore Components of the Fission Yeast γ-Tubulin Complex

Relatively little is known about the in vivo function of individual components of the eukaryotic γ-tubulin complex (γ-TuC). We identified three genes, gfh1+, mod21+, and mod22+, in a screen for fission yeast mutants affecting microtubule organization. gfh1+ is a previously characterized γ-TuC protein weakly similar to human γ-TuC subunit GCP4, whereas mod21+ is novel and shows weak similarity to human γ-TuC subunit GCP5. We show that mod21p is a bona fide γ-TuC protein and that, like gfh1Δ mutants, mod21Δ mutants are viable. We find that gfh1Δ and mod21Δ mutants have qualitatively normal microtubule nucleation from all types of microtubule-organizing centers (MTOCs) in vivo but quantitatively reduced nucleation from interphase MTOCs, and this is exacerbated by mutations in mod22+. Simultaneous deletion of gfh1p, mod21p, and alp16p, a third nonessential γ-TuC protein, does not lead to additive defects, suggesting that all three proteins contribute to a single function. Coimmunoprecipitation experiments suggest that gfh1p and alp16p are codependent for association with a small “core” γ-TuC, whereas mod21p is more peripherally associated, and that gfh1p and mod21p may form a subcomplex independently of the small γ-TuC. Interestingly, sucrose gradient analysis suggests that the major form of the γ-TuC in fission yeast may be a small complex. We propose that gfh1p, mod21p, and alp16 act as facultative “noncore” components of the fission yeast γ-TuC and enhance its microtubule-nucleating ability

Microtubule Cytoskeleton Remodeling by Acentriolar Microtubule-organizing Centers at the Entry and Exit from Mitosis in Drosophila Somatic Cells

Cytoskeleton microtubules undergo a reversible metamorphosis as cells enter and exit mitosis to build a transient mitotic spindle required for chromosome segregation. Centrosomes play a dominant but dispensable role in microtubule (MT) organization throughout the animal cell cycle, supporting the existence of concurrent mechanisms that remain unclear. Here we investigated MT organization at the entry and exit from mitosis, after perturbation of centriole function in Drosophila S2 cells. We found that several MTs originate from acentriolar microtubule-organizing centers (aMTOCs) that contain γ-tubulin and require Centrosomin (Cnn) for normal architecture and function. During spindle assembly, aMTOCs associated with peripheral MTs are recruited to acentriolar spindle poles by an Ncd/dynein-dependent clustering mechanism to form rudimentary aster-like structures. At anaphase onset, down-regulation of CDK1 triggers massive formation of cytoplasmic MTs de novo, many of which nucleated directly from aMTOCs. CDK1 down-regulation at anaphase coordinates the activity of Msps/XMAP215 and the kinesin-13 KLP10A to favor net MT growth and stability from aMTOCs. Finally, we show that microtubule nucleation from aMTOCs also occurs in cells containing centrosomes. Our data reveal a new form of cell cycle–regulated MTOCs that contribute for MT cytoskeleton remodeling during mitotic spindle assembly/disassembly in animal somatic cells, independently of centrioles