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.

Abstract.We have previously shown that a fluorescent derivative of ceramide, N-(e-7-nitrobenz-2-oxa-l,3- diazol-4-yl-aminocaproyl)-D-erythro-sphingosine (C6-NBD-Cer), vitally stains the Golgi apparatus of cells (Lipsky, N. G., and R. E. Pagano. 1985. Science (Wash. DC). 228:745-747). In the present paper we demonstrate that C6-NBD-Cer also accumulates at the Golgi apparatus of fixed cells and we explore the mechanism by which this occurs. When human skin fibroblasts were fixed with glutaraldehyde and then incubated with C6-NBD-Cer at 2°C, the fluorescent lipid spontaneously transferred into the cells, labeling the Golgi apparatus as well as other intracellular membranes. Subsequent incubations with defatted BSA at 24°C removed excess C6-NBD-Cer from the cells such that fluorescence was then detected only at the Golgi apparatus. Similar results were obtained using other cell types. A method for visualizing the fluorescent lipid at the electron microscopic level, based on the photoconversion of a fluorescent marker to a diaminobenzidine product (Sandell, J. H., and R. H. Masland. 1988. J. Histochem. Cytochem.36:555-559), is described and evidence is presented that C6-NBD-Cer was localized to the trans cisternae of the Golgi apparatus. While accumulation occurred in cells fixed in various ways, it was inhibited when fixation protocols that extract or modify cellular lipids were used. In addition, Filipin, which forms complexes with cellular cholesterol, labeled the Golgi apparatus of fixed cells and inhibited accumulation of C6-NBD-Cer at the Golgi apparatus. These results are discussed in terms of a simple model based on the physical properties of C6-NBD-Cer and its interactions with endogenous lipids of the Golgi apparatus. Possible implications of these findings for metabolism and transport of (fluorescent) sphingolipids in vivo are also presented.

The γ-tubulin ring complex (γTuRC) is important for microtubule nucleation from the centrosome. In addition to γ-tubulin, the Drosophila γTuRC contains at least six subunits, three of which [Drosophila gamma ring proteins (Dgrips) 75/d75p, 84, and 91] have been characterized previously. Dgrips84 and 91 are present in both the small γ-tubulin complex (γTuSC) and the γTuRC, while the remaining subunits are found only in the γTuRC. To study γTuRC assembly and function, we first reconstituted γTuSC using the baculovirus expression system. Using the reconstituted γTuSC, we showed for the first time that this subcomplex of the γTuRC has microtubule binding and capping activities. Next, we characterized two new γTuRC subunits, Dgrips128 and 163, and showed that they are centrosomal proteins. Sequence comparisons among all known γTuRC subunits revealed two novel sequence motifs, which we named grip motifs 1 and 2. We found that Dgrips128 and 163 can each interact with γTuSC. However, this interaction is insufficient for γTuRC assembly.

Abstract. We examined the uptake and intracellular transport of the fluorescent glucosylceramide analogue N-[5-(5,7-dimethyl BODIPY'~)-l-pentanoyl] -glucosyl sphingosine (CrDMB-GlcCer) in human skin fibroblasts, and we compared its behavior to that of the corresponding fluorescent analogues of sphingomyelin, galactosylceramide, and lactosylceramide. All four fluorescent analogues were readily transferred from defatted BSA to the plasma membrane during incubation at 4°C. When cells treated with CrDMB-GlcCer were washed, wanned to 370C, and subsequently incubated with defatted BSA to remove fluorescent lipid at the cell surface, strong fluorescence was observed at the Golgi apparatus, as well as weaker labeling at the nuclear envelope and other intracellular membranes. Similar results were obtained with Cs-DMB-galactosylceramide, except that labeling of the Golgi apparatus was weaker than with Cs-DMB-GlcCer. Internalization of Cs-DMB-GlcCer was not inhibited by various treatments, including ATP depletion or wanning to 190C, and biochemical analysis demonstrated that the lipid was not metabolized during its internalization. However, accumulation of Cs-DMB-GlcCer at the Golgi apparatus was reduced when cells were treated with a nonfluorescent analogue of glucosylceramide, suggesting that accumulation of Cs-DMBGlcCer at the Golgi apparatus was a saturable process. In contrast, cells treated with CrDMB-analogues of sphingomyelin or lactosylceramide internalized the fluorescent lipid into a punctate pattern of fluorescence during warming at 37°C, and this process was temperature and energy dependent. These results with CrDMB-sphingomyelin and Cs-DMB-lactosylceramide were analogous to those obtained with another fluorescent analogue of sphingomyelin in which labeling of endocytic vesicles and plasma membrane lipid recycling were documented (Koval, M., and R. E. Pagano. 1990. J. Cell Biol. 111:429--442). Incubation of perforated cells with Cs-DMB-sphingomyelin resulted in prominent labeling of the nuclear envelope and other intracellular membranes, similar to the pattern observed with CrDMB-GIcCer in intact cells. These observations are consistent with the transbilayer movement of fluorescent analogues of glucosylceramide and galactosylceramide at the plasma membrane and early endosomes of human skin fibroblasts, and suggest that both endocytic and nonendocytic pathways are used in the internalization of these lipids from the plasma membrane. Cs-DMB-GaiCer, Cs-DMB-galactosyl sphingosine; Cs-DMB-GlcCer, Cs-DMB-glucosyl sphlngosine; Cs-DMBLa¢Cer, Cs-DMB-lactosyl sphingosine; Cs-DMB-SM, C~-DMB-sphingosylphosphorylcholine; Ct-GalCer, N-hexanoyl-galactosyl sphingosine; Ct-GIcCer, N-hexanoyl-glucosyl sphingosine; amino] hexanoyl])glucosyl sphingosine; Cer, ceramide; GlcCer, glucosylceramide; DF-BSA, defatted BSA; GSL, glycosphingolipid; HMEMB, 10 mM 4-(2-hydroxyethyl)-l-piperazineethane sulfi3aic acid-buffered MEM, pH 7.4, without indicator, and containing 0.5 mM each choline chloride, ethanolamine, serine, and (myo)inositol;...

We have previously shown that when cultured fibroblasts are briefly incubated at 2 degrees C with a fluorescent (NBD) analogue of ceramide, N-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-epsilon-aminohexanoyl]-D-e rythro- sphingosine, fluorescent labeling of the mitochondria, endoplasmic reticulum, and nuclear envelope occurs. During further incubation at 37 degrees C, the Golgi apparatus and later the plasma membrane become intensely fluorescent. Concomitantly, the fluorescent ceramide is metabolized to fluorescent analogues of sphingomyelin and glucosylceramide [Lipsky, N. G., & Pagano, R. E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2608-2612]. In the present study we synthesized fluorescent N-acylsphingosine analogues using various long-chain bases (D-erythro-sphingosine, L-erythro-sphingosine, D-threo-sphingosine, L-threo-sphingosine, D-erythro-dihydrosphingosine, L-threo-dihydrosphingosine, phytosphingosine, and 3-ketosphingosine) and fluorescent fatty acids (epsilon-NBD-aminohexanoic acid; D- or L-alpha-OH-epsilon-NBD-aminohexanoic acid; D- or L-alpha-NBD-aminohexanoic acid). Using previously described resonance energy transfer assays, we examined the rates of spontaneous transfer of these compounds between liposomes and their ability to undergo transbilayer movement. The fluorescent N-acylsphingosine analogues had half-times for spontaneous transfer of 0.3-4.0 min at 25 degrees C, and all were capable of transbilayer movement in lipid vesicles. The metabolism and intracellular distribution of analogues in cultured fibroblasts were also studied. While most of the fluorescent N-acylsphingosines were significantly metabolized to the corresponding sphingomyelin analogues, metabolism to glucosylceramide was strongly dependent on the long-chain base and the stereochemistry of the fluorescent fatty acid moiety.(ABSTRACT TRUNCATED AT 250 WORDS)

Previous studies indicate that γ tubulin ring complex (γTuRC) can nucleate microtubule assembly and may be important in centrosome formation. γTuRC contains approximately eight subunits, which we refer to as Xenopus gamma ring proteins (Xgrips), in addition to γ tubulin. We found that one γTuRC subunit, Xgrip109, is a highly conserved protein, with homologues present in yeast, rice, flies, zebrafish, mice, and humans. The yeast Xgrip109 homologue, Spc98, is a spindle–pole body component that interacts with γ tubulin. In vertebrates, Xgrip109 identifies two families of related proteins. Xgrip109 and Spc98 have more homology to one family than the other. We show that Xgrip109 is a centrosomal protein that directly interacts with γ tubulin. We have developed a complementation assay for centrosome formation using demembranated Xenopus sperm and Xenopus egg extract. Using this assay, we show that Xgrip109 is necessary for the reassembly of salt-disrupted γTuRC and for the recruitment of γ tubulin to the centrosome. Xgrip109, therefore, is essential for the formation of a functional centrosome.