The Role of γ-Tubulin in Centrosomal Microtubule Organization

Eileen O'Toole,Thomas Müller-Reichert,Garrett Greenan,Karen I Lange,Martin Srayko,Adam I Marcus

doi:10.1371/journal.pone.0029795

Abstract

As part of a multi-subunit ring complex, γ-tubulin has been shown to promote microtubule nucleation both in vitro and in vivo, and the structural properties of the complex suggest that it also seals the minus ends of the polymers with a conical cap. Cells depleted of γ-tubulin, however, still display many microtubules that participate in mitotic spindle assembly, suggesting that γ-tubulin is not absolutely required for microtubule nucleation in vivo, and raising questions about the function of the minus end cap. Here, we assessed the role of γ-tubulin in centrosomal microtubule organisation using three-dimensional reconstructions of γ-tubulin-depleted C. elegans embryos. We found that microtubule minus-end capping and the PCM component SPD-5 are both essential for the proper placement of microtubules in the centrosome. Our results further suggest that γ-tubulin and SPD-5 limit microtubule polymerization within the centrosome core, and we propose a model for how abnormal microtubule organization at the centrosome could indirectly affect centriole structure and daughter centriole replication.

Highlights

In most animal cells, the centrosome is the major microtubuleorganizing center (MTOC)
Our observations suggest that c-tubulin is important for the proper microtubule end-morphology and endplacement in the periphery of the centrosome (Figure 6E)
In C. elegans, SPD-5 is required for centrosome structure and recruitment of TBG-1 and other factors [24]

Summary

Introduction

The centrosome is the major microtubuleorganizing center (MTOC). A linescan through the most concentrated mass of microtubules within a single confocal plane in the tbg1(RNAi) embryos indicated that a-tubulin was not excluded in a similar pattern (Figure 1B–C) These results suggested that ctubulin provides a microtubule-organizing function for the centrosome that includes the formation of a microtubule-free zone within the central core, consistent with previous reports [18]. Only 30% (456/1494) of the pole-proximal ends were closed in tbg-1(RNAi) embryos and the rest (1038/1494) were open (Table 1 and Figure S1 for other examples) These results suggest that c-tubulin contributes to the closed-end morphology of microtubules at the centrosome. Tomograms of tbg-1(RNAi) embryos revealed a consistent centriole defect whereby varying numbers of centriolar microtubules extended beyond the distal end of the central tube (Figure 4A–C; Movie S3; Figure S1 for more examples). The daughter central tubes were shorter in spd-5(RNAi) embryos than in wild type (Figure 6D)

Discussion

Materials and Methods