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Microtubule nucleation from centrosomes involves a lockwasher-shaped protein complex containing gamma-tubulin, named the gamma-tubulin ring complex (gammaTuRC). Here we investigate the mechanism by which the gammaTuRC nucleates microtubules, using a direct labelling method to visualize the behaviour of individual gammaTuRCs. A fluorescently-labelled version of the gammaTuRC binds to the minus ends of microtubules nucleated in vitro. Both gammaTuRC-mediated nucleation and binding of the gammaTuRC to preformed microtubules block further minus-end growth and prevent microtubule depolymerization. The gammaTuRC therefore acts as a minus-end-capping protein, as confirmed by electron-microscopic examination of gold-labelled gammaTuRCs. These data support a nucleation model for gammaTuRC function that involves capping of microtubules

The organization and dynamics of microtubules (MTs) are essential for different cellular processes such as migration or division. In animal cells, MT nucleation usually occurs at the centrosome, where γ-tubulin plays a key role. This protein is organized in multiprotein complexes (Moritz et al., 1995; Zheng et al., 1995; Raynaud-Messina and Merdes, 2007). In Drosophila melanogaster, two main complexes have been characterized (Oegema et al., 1999). The γ-tubulin small complex (γ-TuSC) is composed of γ-tubulin and two other proteins, Dgrip84 and -91. The γ-tubulin ring complex (γ-TuRC) is formed of γ-TuSCs associated with additional grip motif polypeptides, Dgrip75, -128, and -163, and a WD motif protein, Dgp71WD. Components of the γ-TuSC are highly conserved in eukaryotes. Deletion of any γ-TuSC subunit is lethal both in fungi and in Drosophila. This loss of function results in the accumulation of cells in mitosis, which is associated with defects such as monopolar spindles, impairment in centrosome maturation, and aneuploidy (Oakley and Oakley, 1989; Sunkel et al., 1995; Knop and Schiebel, 1997; Barbosa et al., 2000; Colombié et al., 2006). In contrast, γ-TuRC–specific grip motif proteins are nonessential for viability in yeast and in Drosophila (Fujita et al., 2002; Schnorrer et al., 2002; Anders et al., 2006; Vérollet et al., 2006; Vogt et al., 2006). Nevertheless, these grip proteins are necessary for the assembly of the large complex, for efficient mitotic progression (Vérollet et al., 2006; Izumi et al., 2008), and for specialized functions such as the organization of MT subarrays during oogenesis (Schnorrer et al., 2002; Vogt et al., 2006). Analysis of the nongrip component Dgp71WD reveals that this protein regulates the function and targeting of the γ-TuRC to the centrosome and along spindle MTs (Haren et al., 2006; Lüders et al., 2006).

The γ-tubulin complexes are involved in the nucleation of MTs from centrosomes but also from chromatin and spindle MTs (Joshi et al., 1992; Sunkel et al., 1995; Knop and Schiebel, 1997; Oegema et al., 1999; Wilde and Zheng, 1999; Wiese and Zheng, 2000; Goshima et al., 2008; Zhu et al., 2008). Additional observations in fungi suggest that γ-tubulin and its partners also affect the organization or dynamics of MTs (Oakley and Oakley, 1989; Paluh et al., 2000; Vardy and Toda, 2000; Fujita et al., 2002; Venkatram et al., 2004; Zimmerman and Chang, 2005; Masuda et al., 2006). To determine whether and how γ-TuRC proteins could influence MT dynamics, we determined dynamic parameters on individual MTs in Drosophila S2 cells during interphase. For the first time in metazoan cells, we show that γ-TuRCs contribute to the regulation of MT dynamics, independently of their nucleation activity. The γ-TuRCs localize along MTs, where they limit catastrophe events, thus enhancing MT stability.

Tubulin is a small globular protein found in all eukaryotic cells. The tubulin family represents about 3-4% of the total protein content in a cell and its members include α-, β-, γ-, δ-, ε-, and ζ-tubulin [1]. Although the different forms of tubulin are similar, they can have different cellular locations and functions. It should be noted that although both actin and tubulin are basic components of the cytoskeleton and possess the ability to hydrolyze NTP, they are evolutionarily distinct with actin being related in structure to hexokinase and tubulins being distantly related to heterotrimeric G-proteins and other GTPases such as Ras [2].

[pic]Figure 1. y-TURC

Repeating units of ~110kDa α/β-tubulin heterodimers are the primary component of microtubules. Microtubule assembly starts with the assembly of a tubulin complex. Here, γ-tubulin and other protein components form the γ-tubulin ring complex (γ-TuRC) [2]. The specific cellular roles for additional tubulins such as δ-, ε- and ζ- tubulins are less well known, but they can be found in centrioles and may be involved during mitotic spindle assembly and cell division [3]. Post-translational modification of tubulin such as acetylation, detyrosination, phosphorylation etc. have also been described at length [4]. These modifications may influence the structural properties of microtubules, alter their dynamics and affect how they function within the cell.

Nucleation of Tubulin subunits[Edit]

Microtubule nucleation is unfavorable under normal conditions found in most living cells. Consequently, microtubules are nucleated from a complex of γ-tubulin and other protein components known as the γ-tubulin ring complex (γ-TuRC). γ-TuRC nucleates and caps the minus end of new filaments by providing stable binding sites for tubulin dimers [5]. Tubulin dimers primarily use longitudinal interactions to bind to each other and to γ−TuRC during the nucleation phase. As the protofilament length increases, lateral interactions between the protofilaments create additional stability that leads to a closed microtubule [2].

[pic]Figure 2. Structure of the centrosome: In non-dividing cells, the centrosome, which is also known as the MTOC, consists of a pair of L-shaped centrioles and associated pericentriolar material. The 'older' of the two centrioles has additional proteins that form appendages along the exterior surface. The pericentriolar material contains numerous γ-TuRCs that nucleate the microtubule array. The centrioles have microtubules organized in a structure similar to the basal bodies found at the base of cilia and flagella. Figure adapted from [5]

After the slow nucleation phase, microtubules elongate rapidly. The minus end of γ-tubulin is anchored near the MTOC, whilst the plus end of γ-tubulin is exposed. This allows elongation to occur from the exposed γ-tubulin through interactions with the minus end of α/β-tubulin heterodimers [2]. Although it remains unclear whether the formation of longitudinal contacts with α-tubulin stimulates γ-tubulin hydrolysis of GTP, the rate of GTP hydrolysis on β-tubulin, along with its concentration are determining factors of microtubule assembly [2]. Once a tubulin dimer has been added to the lattice, the more likely it is for the GTP on β-tubulin to be hydrolyzed. MAPs can also control the rate of assembly/disassembly, the rate of GTP hydrolysis and the overall length of microtubules.

References

1. An abundance of tubulins. Trends Cell Biol. 2000; 10(12). [PMID: 11121746] 2. Kollman JM., Merdes A., Mourey L., Agard DA. Microtubule nucleation by γ-tubulin complexes. Nat. Rev. Mol. Cell Biol. 2011; 12(11). [PMID: 21993292] 3. McKean PG., Vaughan S., Gull K. The extended tubulin superfamily. J. Cell. Sci. 2001; 114(Pt 15). [PMID: 11683407] 4. Janke C., Bulinski JC. Post-translational regulation of the microtubule cytoskeleton: mechanisms and functions. Nat. Rev. Mol. Cell Biol. 2011; 12(12). [PMID: 22086369] 5. Wiese C., Zheng Y. A new function for the gamma-tubulin ring complex as a microtubule minus-end cap. Nat. Cell Biol. 2000; 2(6). [PMID: 10854327]