Reconstitution of centrosome microtubule nucleation in Spisula

Evidence strongly supports the hypothesis that gamma-TuRCs are sites for the nucleation of microtubules within the centrosome PCM (Zheng et al., 1995; Moritz et al., 1995a,b). Further, these structures appear to be recruited to the centrosome from cytoplasmic pools during centrosome assembly and centrosome maturation (Moritz et al., 1998; Schnackenberg et al., 1998, 2000; Schnackenberg and Palazzo, 1999; Khodjakov and Rieder, 1999). It has also been shown that the centrosome PCM contains a network of filamentous fibers, which function as a scaffold for binding these microtubule nucleating sites (Schnackenberg et al., 1998). However, binding gamma-tubulin rings to this scaffold requires at least one additional factor (Moritz et al., 1998; Schnackenberg et al., 2000). Because extracts prepared from a variety of biological sources are capable of supporting the recovery of microtubule nucleation by Spisula KICRs (Schnackenberg et al., 2000), the methods outlined in this chapter could prove useful in the search for factors that are necessary for centrosome assembly and the increase in centrosome-dependent microtubule nucleation during centrosome maturation.