GCP-WD is a γ-tubulin targeting factor required for centrosomal and chromatin-mediated microtubule nucleation

Abstract

The gamma-tubulin ring complex (gammaTuRC) is a large multi-protein complex that is required for microtubule nucleation from the centrosome. Here, we show that the GCP-WD protein (originally named NEDD1) is the orthologue of the Drosophila Dgrip71WD protein, and is a subunit of the human gammaTuRC. GCP-WD has the properties of an attachment factor for the gammaTuRC: depletion or inhibition of GCP-WD results in loss of the gammaTuRC from the centrosome, abolishing centrosomal microtubule nucleation, although the gammaTuRC is intact and able to bind to microtubules. GCP-WD depletion also blocks mitotic chromatin-mediated microtubule nucleation, resulting in failure of spindle assembly. Mitotic phosphorylation of GCP-WD is required for association of gamma-tubulin with the spindle, separately from association with the centrosome. Our results indicate that GCP-WD broadly mediates targeting of the gammaTuRC to sites of microtubule nucleation and to the mitotic spindle, which is essential for spindle formation.

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

“…GCP5 and GCP6 are also present in stoichiometric amounts, but about three times less abundant than GCP2 or GCP3, and GCP4 is nearly twice as abundant as GCP5 and GCP6, but only half as abundant as GCP2 and GCP3. Finally, three proteins associated with the γ‐TuRC, Nedd1 (Haren et al , 2006; Luders et al , 2006) and Mozart 1, and Mozart 2 (Hutchins et al , 2010; Teixido‐Travesa et al , 2010) are similarly abundant as the GCP2 and GCP3 core components of γ‐TuSC. These data extend previous biochemical studies (Murphy et al , 2001; Choi et al , 2010) and support the notion that multiple γ‐TuSCs associate with GCP4, GCP5, and GCP6 into γ‐TuRCs (Kollman et al , 2011; Lin et al , 2015).…”

Generation of noncentrosomal microtubule arrays

“…also known as NEDD1) that appears to tether ␥-TuRCs at the centrosome without being necessary for the integrity of the ␥-TuRC, and this would be an obvious candidate for sites of regulation and release of MTs with bound ␥-TuRCs (Luders et al, 2006). Despite the attractiveness of this hypothesis, there is little indication that ␥-tubulin is present on minus ends of stable noncentrosomal MTs, and in neurons there is evidence that is not (Baas and Joshi, 1992).…”

“…␥-TuRCs are highly conserved structures composed of ␥-tubulin and associated proteins, and in fission yeast all ␥-tubulin complex (␥-TuC) components are needed for cytoplasmic MT organization (see Weise and Zheng, 2006, in this issue). Other proteins, such as pericentrin, NEDD1 and ninein contribute to centrosome function by recruiting and tethering ␥-TuRCs to the centrosome and anchoring newly formed MTs (Dictenberg et al, 1998;Luders et al, 2006;Mogensen et al, 2000). Typically, interphase cells have a single centrosomal array, whereas mitotic cells have two centrosomal arrays.…”

Kinesin-14 Family Proteins HSET/XCTK2 Control Spindle Length by Cross-Linking and Sliding Microtubules

“…Immunoprecipitation in HeLa extract was performed as described (Luders et al, 2006) using anti-HSET antibodies cross-linked to Affi-prep protein A beads. The GFP-HSET overexpressing HeLa cells were plated in a 10-cm plate and treated with 100 ng/ml nocodazole overnight.…”