Centrosome assembly in vitro: role of gamma-tubulin recruitment in Xenopus sperm aster formation

Abstract

Centrioles organize microtubules in two ways: either microtubules elongate from the centriole cylinder itself, forming a flagellum or a cilium ("template elongation"), or pericentriolar material assembles and nucleates a microtubule aster ("astral nucleation"). During spermatogenesis in most species, a motile flagellum elongates from one of the sperm centrioles, whereas after fertilization a large aster of microtubules forms around the sperm centrioles in the egg cytoplasm. Using Xenopus egg extracts we have developed an in vitro system to study this change in microtubule-organizing activity. An aster of microtubules forms around the centrioles of permeabilized frog sperm in egg extracts, but not in pure tubulin. However, when the sperm heads are incubated in the egg extract in the presence of nocodazole, they are able to nucleate a microtubule aster after isolation and incubation with pure calf brain tubulin. This provides a two-step assay that distinguishes between centrosome assembly and subsequent microtubule nucleation. We have studied several centrosomal antigens during centrosome assembly. The CTR2611 antigen is present in the sperm head in the peri-centriolar region. gamma-tubulin and certain phosphorylated epitopes appear in the centrosome only after incubation in the egg extract. gamma-tubulin is recruited from the egg extract and associated with electron-dense patches dispersed in a wide area around the centrioles. Immunodepletion of gamma-tubulin and associated molecules from the egg extract before sperm head incubation prevents the change in microtubule-organizing activity of the sperm heads. This suggests that gamma-tubulin and/or associated molecules play a key role in centrosome formation and activity.

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

“…Microtubule aster formation on nascent centrosomes of sperm nuclei in Xenopus extracts is dependent on the recruitment of soluble ␥ TuRCs to these sites (Felix et al, 1994;Stearns and Kirschner, 1994). Previous studies implicated pericentrin in this process (Dictenberg et al, 1998;Doxsey et al, 1994).…”

“…The mechanism of aster inhibition was examined in more detail by monitoring recruitment of ␥ tubulin onto nascent centrosomes in Xenopus mitotic extracts as described previously (Doxsey et al, 1994;Felix et al, 1994;Stearns and Kirschner, 1994). The pericentrin C-terminal domain and subdomains of this protein specifically inhibited recruitment of ␥ tubulin onto centrosomes to the same extent and at the same concentration that prevented microtubule aster assembly and disrupted the interaction between pericentrin and the ␥ TuRC (Figure 4, E-G).…”

Xenopus TACC3/Maskin Is Not Required for Microtubule Stability but Is Required for Anchoring Microtubules at the Centrosome

“…To test this idea, we developed what we named the sequential assay. In this assay, salt-stripped centrosomes were first incubated with maskin-depleted extracts (presumably to rebuild centrosome substructure and recruit ␥-tubulin, without which centrosomes are nonfunctional (Felix et al, 1994;Moritz et al, 1998;Schnackenberg et al, 1998). The extract was then removed, and, after a buffer wash step, the centrosomes were incubated with recombinant maskin or maskin truncation mutants in buffer (Figure 4).…”

Reconstitution and Characterization of Budding Yeast γ-Tubulin Complex

“…Centrosome-dependent microtubule polymerization in higher eukaryotes involves recruitment of the ␥-TuRC to the centrosome and activation of the nucleation machinery that results in aster formation (Felix et al, 1994;Stearns and Kirschner, 1994;Moritz et al, 1998;Schnackenberg et al, 1998). Studies in Drosophila indicate that although the large ␥-TuRC has robust nucleating activity in vitro, the ␥-TuSC has weak activity (Oegema et al, 1999;Gunawardane et al, 2000).…”