PERICENTRIN AND GAMMA-TUBULIN FORM A PROTEIN COMPLEX AND ARE ORGANIZED INTO A NOVEL LATTICE AT THE CENTROSOME

Pericentrin and gamma-tubulin are integral centrosome proteins that pl ay a role in microtubule nucleation and organization. In this study, w e examined the relationship between these proteins in the cytoplasm an d at the centrosome, In extracts prepared from Xenopus eggs, the prote ins were part of a large complex as demonstrated by sucrose gradient s edimentation, gel filtration and coimmunoprecipitation analysis. The p ericentrin-gamma-tubulin complex was distinct from the previously desc ribed gamma-tubulin ring complex (gamma-TuRC) as purified gamma-TuRC f ractions did not contain detectable pericentrin. When assembled at the centrosome, the two proteins remained in close proximity as shown by fluorescence resonance energy transfer. The three-dimensional organiza tion of the centrosome-associated fraction of these proteins was deter mined using an improved immunofluorescence method. This analysis revea led a novel reticular lattice that was conserved from mammals to amphi bians, and was organized independent of centrioles. The lattice change d dramatically during the cell cycle, enlarging from G1 until mitosis, then rapidly disassembling as cells exited mitosis, In cells colabele d to detect centrosomes and nucleated microtubules, lattice elements a ppeared to contact the mi nus ends of nucleated microtubules. Our resu lts indicate that pericentrin and gamma-tubulin assemble into a unique centrosome lattice that represents the higher-order organization of m icrotubule nucleating sites at the centrosome.