Spindle Dynamics and the Role of γ-Tubulin in EarlyCaenorhabditis elegansEmbryos

Abstract

γ-Tubulin is a ubiquitous and highly conserved component of centrosomes in eukaryotic cells. Genetic and biochemical studies have demonstrated that γ-tubulin functions as part of a complex to nucleate microtubule polymerization from centrosomes. We show that, as in other organisms, Caenorhabditis elegans γ-tubulin is concentrated in centrosomes. To study centrosome dynamics in embryos, we generated transgenic worms that express GFP::γ-tubulin or GFP::β-tubulin in the maternal germ line and early embryos. Multiphoton microscopy of embryos produced by these worms revealed the time course of daughter centrosome appearance and growth and the differential behavior of centrosomes destined for germ line and somatic blastomeres. To study the role of γ-tubulin in nucleation and organization of spindle microtubules, we used RNA interference (RNAi) to deplete C. elegansembryos of γ-tubulin. γ-Tubulin (RNAi) embryos failed in chromosome segregation, but surprisingly, they contained extensive microtubule arrays. Moderately affected embryos contained bipolar spindles with dense and long astral microtubule arrays but with poorly organized kinetochore and interpolar microtubules. Severely affected embryos contained collapsed spindles with numerous long astral microtubules. Our results suggest that γ-tubulin is not absolutely required for microtubule nucleation in C. elegans but is required for the normal organization and function of kinetochore and interpolar microtubules.

Mitosis-specific Anchoring of γ Tubulin Complexes by Pericentrin Controls Spindle Organization and Mitotic Entry

“…Under these conditions, centrosomes in Caenorhabditis elegans and Drosophila embryos were compromised in their ability to form mitotic asters (Hannak et al, 2002;Strome et al, 2001), separate from one another (Barbosa et al, 2003;Sampaio et al, 2001), and organize meiotic and mitotic spindles (Sunkel et al, 1995;Barbosa et al, 2000Barbosa et al, , 2003. It is of interest that mitotic asters in some of these systems formed in the absence of ␥ tubulin or other ␥ tubulin ring complex proteins (Strome et al, 2001;Hannak et al, 2002;Barbosa et al, 2003). This is in contrast to our results in Xenopus extracts where microtubule asters did not form in the presence of the pericentrin interacting domain of GCP2/3 even after extended periods (30 min).…”

“…Within the matrix are large protein complexes of ␥ tubulin and associated proteins that have a ring-like structure and mediate the nucleation of microtubules called ␥ tubulin ring complexes or ␥ TuRCs (Moritz et al, 1995a;Zheng et al, 1995). Other proteins may share the ability to nucleate microtubules because centrosomes can organize microtubules in the absence of functional ␥ tubulin (Sampaio et al, 2001;Strome et al, 2001;Hannak et al, 2002).…”

“…Within the matrix are large protein complexes of ␥ tubulin and associated proteins that have a ring-like structure and mediate the nucleation of microtubules called ␥ tubulin ring complexes or ␥ TuRCs (Moritz et al, 1995a;Zheng et al, 1995). Other proteins may share the ability to nucleate microtubules because centrosomes can organize microtubules in the absence of functional ␥ tubulin (Sampaio et al, 2001;Strome et al, 2001;Hannak et al, 2002).During cell cycle progression, centrosomes "mature" by recruiting additional ␥ TuRCs and several other proteins, resulting in an increase in the nucleation capacity of the centrosome (reviewed in Blagden and Glover, 2003). However, we still know very little about proteins that directly anchor ␥ TuRCs to centrosomes in vertebrate cells.…”

Characterization of a New γTuRC Subunit with WD Repeats

“…This led to the hypothesis that ␥-tubulin is involved in microtubule nucleation at the centrosomes. Recent studies of ␥-tubulin in Schizosaccharomyces pombe, Caenorhabditis elegans, and Aspergillus nidulans suggest that ␥-tubulin is also involved in MT organization and spindle assembly (Paluh et al, 2000;Prigozhina et al, 2001;Strome et al, 2001). Therefore, ␥-tubulin appears to have roles in MT organization in addition to regulating microtubule nucleation.…”

A Spindle Checkpoint Functions during Mitosis in the EarlyCaenorhabditis elegansEmbryo

“…GFP expressed in ␤-tubulin labels microtubules and centrosomes (Praitis et al, 2001); pJH4.52: his-11::GFP. GFP expressed in H2B histones throughout the cell cycle (Strome et al, 2001).apo-5(or358ts) and dnc-1(or404ts) were isolated in a screen for temperaturesensitive (ts) maternal-effect embryonic-lethal mutations, using methods described previously (Encalada et al, 2000). Both or358 and or404 were backcrossed five times by using either him-8 or N2 males.…”