Although Kendrin and CG-NAP are good candidates for tethering γTu RC to the centrosome, it remains unclear whether they are essential for centrosome-mediated microtubule nucleation.
For example, one study showed that depleting Kendrin did not affect the ability of Chinese hamster ovary cell lysates to complement KI-extracted centrosomes (Li , 1 m M EGTA, and 1 m M β-mercaptoethanol; HB100Na, HB buffer containing 100 m M Na Cl; HB100K, HB buffer containing 100 m M KCl; and HB3, HB buffer containing 100 m M KCl, 10% glycerol, protease inhibitor (0 dilution from protease inhibitor stock), 1 m M phenylmethylsulfonyl fluoride.
Briefly, the isolated centrosomes were treated with 2 M KI on ice for 7 min.
The column was washed with HB100Na, and bound proteins were eluted by a linear Na Cl gradient (0.1–0.5 M).
Embryo extracts or protein fractions were used to complement the salt-extracted centrosome scaffolds to assemble into functional centrosomes according to published methods with minor modifications (Moritz ., 1998).
The centrosome in animal cells provides a major microtubule-nucleating site that regulates the microtubule cytoskeleton temporally and spatially throughout the cell cycle.
We report the identification in k Da (CP309), cofractionates with the γ-tubulin ring complex and the centrosome-complementing activity.
Sucrose gradients (5–40%) were prepared as described previously in HB100K containing 0.1 m M GTP and 0 protease inhibitor stock (Oegema ., 2001).
The resuspended and clarified PEG-precipitated proteins were loaded onto the gradient and centrifuged in a SW55 rotor at 50,000 rpm for 4 h at 4°C. Fractions 11–13 were combined, filtered, and subjected to further purification on a Mono S column (HR 5/5; Pharmacia Amersham Biosciences, Piscataway, NJ) equilibrated with HB buffer.
The mixture was incubated on ice for 20 min and centrifuged at 14,000 rpm for 15 min at 4°C.
The precipitant was resuspended with HB100K containing 0.1 m M GTP and 0.05% NP-40 in 1/4 volume of the clarified extract, homogenized, and centrifuged at 14,000 rpm for 15 min at 4°C.
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.
Interestingly, Spc110 contains a calmodulin (Ca M)-binding motif at its C terminus.