The microtubule cytoskeleton is essential for a variety of cellular processes, including cell movement, organelle transport, and cell division.The centrosome, consisting of a pair of centrioles and a pericentriolar material, provides the major microtubule-nucleating function in animal cells (Kellogg ., 1994).Thus far, other than the mammalian centriolar tubulin, only one mammalian centrosome protein, the outer dense fiber 2, has been shown to remain in the centrosome scaffold after high salt extraction (Nakagawa ., 1998), two similar assays have been developed to study the microtubule-nucleating activity of the centrosome.
Briefly, the isolated centrosomes were treated with 2 M KI on ice for 7 min.
The KI-stripped centrosomes were first attached to the polylysine-coated coverslips and then incubated with 60 μl of embryo extract or 60 μl of different fractions from PEG precipitation, sucrose gradient sedimentation, or Mono S column chromatography for 15 min at 30°C.
By analogy, these studies suggest that Ca M and Ca M-binding proteins might be required for centrosome-mediated microtubule nucleation by tethering γTu RC to centrosomes in animal cells.
Consistent with this idea, two mammalian centrosome proteins, Kendrin (also called pericentrin B) and CG-NAP (also called AKAP350 or AKAP450), were found to contain Ca M binding motifs homologous to Spc110 (Takahashi ., 2002).
The centrosome proteins can also be classified based on whether microtubules are required for their localization to the centrosomes.
γTu RC belongs to the group of centrosome proteins whose centrosomal localization is independent of microtubules.
Protease inhibitor stock contained 10 m M benzamidine-HCl, 0.1 mg/ml phenanthroline, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin A in ethanol.
Polyethylene glycol 8000 (PEG8000) was added to the clarified embryo extract to a final concentration of 3% from a 30% stock in HB100Na.
After incubation, soluble proteins were washed out and then a mixture of unlabeled and rhodamine-labeled tubulin was added and incubated for 10 min at 30°C.
The number of microtubule asters nucleated from the reconstituted centrosomes was counted under a fluorescence microscope using a 60× objective.
We show that CP309 is required for microtubule nucleation mediated by centrosomes and that it interacts with the γ-tubulin small complex.