The kinetically dominant assembly pathway for centrosomal asters in Caenorhabditis elegans is γ-tubulin dependent

Abstract

γ-Tubulin–containing complexes are thought to nucleate and anchor centrosomal microtubules (MTs). Surprisingly, a recent study (Strome, S., J. Powers, M. Dunn, K. Reese, C.J. Malone, J. White, G. Seydoux, and W. Saxton. Mol. Biol. Cell. 12:1751–1764) showed that centrosomal asters form in Caenorhabditis elegans embryos depleted of γ-tubulin by RNA-mediated interference (RNAi). Here, we investigate the nucleation and organization of centrosomal MT asters in C. elegans embryos severely compromised for γ-tubulin function. We characterize embryos depleted of ∼98% centrosomal γ-tubulin by RNAi, embryos expressing a mutant form of γ-tubulin, and embryos depleted of a γ-tubulin–associated protein, CeGrip-1. In all cases, centrosomal asters fail to form during interphase but assemble as embryos enter mitosis. The formation of these mitotic asters does not require ZYG-9, a centrosomal MT-associated protein, or cytoplasmic dynein, a minus end–directed motor that contributes to self-organization of mitotic asters in other organisms. By kinetically monitoring MT regrowth from cold-treated mitotic centrosomes in vivo, we show that centrosomal nucleating activity is severely compromised by γ-tubulin depletion. Thus, although unknown mechanisms can support partial assembly of mitotic centrosomal asters, γ-tubulin is the kinetically dominant centrosomal MT nucleator.

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.…”

Noncentrosomal microtubules in C. elegans epithelia

“…MT minus-end binding proteins display stabilizing or protecting activities, and largely contribute to noncentrosomal MT generation. C. elegans possesses three known -TIPs (Box 1): g-tubulin and its associated proteins GIP-1 and GIP-2 (Hannak et al, 2002), PTRN-1 [belonging to the conserved Patronin/ Nehza/calmodulin and spectrin-associated family (CAMSAP) (Goodwin and Vale, 2010)], and NOCA-1(noncentrosomal array 1) with homology to ninein (Wang et al, 2015). PTRN-1 function has mostly been described in neurons (Marcette et al, 2014;Richardson et al, 2014), and axon regeneration (Chuang et al, 2014).…”

“…They include the major MT nucleator g-tubulin/TBG-1 and its associated complex containing GIP-1 and GIP-2 (gamma-tubulin interacting protein, formerly called CeGrip-1/2, Hannak et al, 2002); -TIPs also include NOCA-1 (Wang et al, 2015) and the patronin homologue PTRN-1, formerly PQN-34 (Goodwin and Vale, 2010; see main text for details).…”