γ-tubulin exists in two related complexes in Drosophila embryo extracts (Moritz, M., Y. Zheng, B.M. Alberts, and K. Oegema. 1998. J. Cell Biol. 142:1– 12). Here, we report the purification and characterization of both complexes that we name γ-tubulin small complex (γTuSC; ∼280,000 D) and Drosophila γTuRC (∼2,200,000 D). In addition to γ-tubulin, the γTuSC contains Dgrip84 and Dgrip91, two proteins homologous to the Spc97/98p protein family. The γTuSC is a structural subunit of the γTuRC, a larger complex containing about six additional polypeptides. Like the γTuRC isolated from Xenopus egg extracts (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578–583), the Drosophila γTuRC can nucleate microtubules in vitro and has an open ring structure with a diameter of 25 nm. Cryo-electron microscopy reveals a modular structure with ∼13 radially arranged structural repeats. The γTuSC also nucleates microtubules, but much less efficiently than the γTuRC, suggesting that assembly into a larger complex enhances nucleating activity. Analysis of the nucleotide content of the γTuSC reveals that γ-tubulin binds preferentially to GDP over GTP, rendering γ-tubulin an unusual member of the tubulin superfamily.

Abstract. A series of ceramide analogues bearing the fluorophore boron dipyrromethene difluoride (BODIPY) were synthesized and evaluated as vital stains for the Golgi apparatus, and as tools for studying lipid traffic between the Golgi apparatus and the plasma membrane of living cells. Studies of the spectral properties of several of the BODIPY-labeled ceramides in lipid vesicles demonstrated that the fluorescence emission maxima were strongly dependent upon the molar density of the probes in the membrane. This was especially evident using N- [5-(5,7-dimethyl (Cs-DMB-Cer), which exhibited a shift in its emission maximum from green (,x,515 nm) to red (•620 nm) wavelengths with increasing concentrations. When C5-DMB-Cer was used to label living cells, this property allowed us to differentiate membranes containing high concentrations of the fluorescent lipid and its metabolites (the corresponding analogues of sphingomyelin and glucosylceramide) from other regions of the cell where smaller mounts of the probe were present. Using this approach, prominent red fluorescent labeling of the Golgi apparatus, Golgi apparatus-associated tubulovesicular processes, and putative Golgi apparatus transport vesicles was seen in living human skin fibroblasts, as well as in other cell types. Based on fluorescence ratio imaging microscopy, we estimate that Cs-DMB-Cer and its metabolites were present in Golgi apparatus membranes at concentrations up to 5-10 mol %. In addition, the concentration-dependent spectral properties of C~-DMB-Cer were used to monitor the transport of Cs-DMB-lipids to the cell surface at 37°C. . This molecule is a vital stain for the Golgi apparatus (24), and, in combination with fluorescence video imaging, has been used to study the dynamics of this organelle within living cells (8). C6-NBD-Cer also stains the Golgi apparatus of fixed cells, most likely through interaction(s) with endogenous lipids and cholesterol, and serves as a trans-Golgi marker for both light and electron microscopy in these preparations (29,33). Similar to its endoge- nous counterpart, C6-NBD-Cer is metabolized in living cells to sphingomyelin (SM) and a glycolipid, glucosylceramide (GlcCer) (17,22,23,32,44,45). These fluorescent metabolites are subsequently transported to the plasma membrane of cells from the Golgi complex by a vesicle-mediated process (17,23). In polarized cells the fluorescent Cer is metabolized to fluorescent analogues of SM and GlcCer, and the latter is preferentially delivered to the apical cell surface (44,45). This polarized delivery is consistent with the known enrichment of glycosphingolipids in apical membranes, and indicates that C6-NBD-Cer and its metabolites are recognized by the cellular sorting and transport machinery in a manner similar to their natural counterparts. BODIPY)-l-pentanoyl]-D-erythro-sphingosineAlthough much useful information has already been obtained using C6-NBD-Cer, there are several disadvantages of this probe. First, the NBD-fluorophore is rapidly bleached during observation under...

The activated form of Ran (Ran-GTP) stimulates spindle assembly in Xenopus laevis egg extracts, presumably by releasing spindle assembly factors, such as TPX2 (target protein for Xenopus kinesin-like protein 2) and NuMA (nuclear-mitotic apparatus protein) from the inhibitory binding of importin-alpha and -beta. We report here that Ran-GTP stimulates the interaction between TPX2 and the Xenopus Aurora A kinase, Eg2. This interaction causes TPX2 to stimulate both the phosphorylation and the kinase activity of Eg2 in a microtubule-dependent manner. We show that TPX2 and microtubules promote phosphorylation of Eg2 by preventing phosphatase I (PPI)-induced dephosphorylation. Activation of Eg2 by TPX2 and microtubules is inhibited by importin-alpha and -beta, although this inhibition is overcome by Ran-GTP both in the egg extracts and in vitro with purified proteins. As the phosphorylation of Eg2 stimulated by the Ran-GTP-TPX2 pathway is essential for spindle assembly, we hypothesize that the Ran-GTP gradient established by the condensed chromosomes is translated into the Aurora A kinase gradient on the microtubules to regulate spindle assembly and dynamics.