In vitro assembly and GTP hydrolysis by bacterial tubulins BtubA and BtubB

Arecent study identified genuine tubulin proteins, BtubA and BtubB, in the bacterial genus Prosthecobacter. We have expressed BtubA and BtubB in Escherichia coli and studied their in vitro assembly. BtubB by itself formed rings with an outer diameter of 35-36 nm in the presence of GTP or GDP. Mixtures of BtubB and BtubA formed long protofilament bundles, 4-7 protofilaments wide (20-30 protofilaments in the three-dimensional bundle). Regardless of the starting stoichiometry, the polymers always contained equal concentrations of BtubA and BtubB, suggesting that BtubA and B alternate along the protofilament. BtubA showed negligible GTP hydrolysis, whereas BtubB hydrolyzed 0.40 mol GTP per min per mol BtubB. This GTPase activity increased to 1.37 per min when mixed 1:1 with BtubA. A critical concentration of 0.4-1.0 microM was indicated by light scattering experiments and extrapolation of GTPase versus concentration, thus suggesting a cooperative assembly mechanism.     

Molecular dynamics modeling of tubulin C-terminal tail interactions with the microtubule surface

Tubulin, an α/β heterodimer, has had most of its 3D structure analyzed; however, the carboxy (C)-termini remain elusive. Importantly, the C-termini play critical roles in regulating microtubule structure and function. They are sites of most of the post-translational modifications of tubulin and interaction sites with molecular motors and microtubule-associated proteins. Simulated annealing was used in our molecular dynamics modeling to predict the interactions of the C-terminal tails with the tubulin dimer. We examined differences in their flexibility, interactions with the body of tubulin, and the existence of structural motifs. We found that the α-tubulin tail interacts with the H11 helix of β-tubulin, and the β-tubulin tail interacts with the H11 helix of α-tubulin. Tail domains and H10/B9 loops interact with each other and compete for interactions with positively-charged residues of the H11 helix on the neighboring monomer. In a simulation in which α-tubulin's H10/B9 loop switches on sub-nanosecond intervals between interactions with the C-terminal tail of α-tubulin and the H11 helix of β-tubulin, the intermediate domain of α-tubulin showed more fluctuations compared to those in the other simulations, indicating that tail domains may cause shifts in the position of this domain. This suggests that C-termini may affect the conformation of the tubulin dimer which may explain their essential function in microtubule formation and effects on ligand binding to microtubules. Our modeling also provides evidence for a disordered-helical/helical double-state system of the T3/H3 region of the microtubule, which could be linked to depolymerization following GTP hydrolysis.     

Glycolytic enzyme–tubulin interactions: Role of tubulin carboxy terminals

Tubulin and microtubules were modified with the protease, subtilisin. The modification reduced the length of α-or β-tubulin by cleaving a peptide fragment from the C-terminals. Generation of α′β′-tubulin, which is cleaved at both the α- and β-subunit terminals, and αβ′-tubulin, which is cleaved at the β′-subunit C-terminal, have already been reported. In this work an isotype, α′β-tubulin, was produced. The three modified tubulin isotypes were compared for their ability to interact with glycolytic enzymes. Cleavage of α led to a poorer interaction when tested via affinity chromatography. Tubulin also inhibits the activity of aldolase and glyceraldehyde 3-phosphate dehydrogenase. When the α-subunit C-terminal was intact, inhibition was greatest. These results imply that the C-terminal of the tubulin α-subunit is subunit is responsible for interactions with glycolytic enzymes.     

The solution structure of the N-terminal domain of human tubulin binding cofactor C reveals a platform for tubulin interaction

Human Tubulin Binding Cofactor C (TBCC) is a post-chaperonin involved in the folding and assembly of α- and β-tubulin monomers leading to the release of productive tubulin heterodimers ready to polymerize into microtubules. In this process it collaborates with other cofactors (TBC's A, B, D, and E) and forms a supercomplex with TBCD, β-tubulin, TBCE and α-tubulin. Here, we demonstrate that TBCC depletion results in multipolar spindles and mitotic failure. Accordingly, TBCC is found at the centrosome and is implicated in bipolar spindle formation. We also determine by NMR the structure of the N-terminal domain of TBCC. The TBCC N-terminal domain adopts a spectrin-like fold topology composed of a left-handed 3-stranded α-helix bundle. Remarkably, the 30-residue N-terminal segment of the TBCC N-terminal domain is flexible and disordered in solution. This unstructured region is involved in the interaction with tubulin. Our data lead us to propose a testable model for TBCC N-terminal domain/tubulin recognition in which the highly charged N-terminus as well as residues from the three helices and the loops interact with the acidic hypervariable regions of tubulin monomers.     

Proteolytic analysis of polymerized maize tubulin: regulation of microtubule stability to low temperature and Ca2+ by the carboxyl terminus of β-tubulin

To obtain information on plant microtubule stability to low temperature and Ca²⁺, the regulatory domain of polymerized tubulin from maize (Zea mays ev. Black Mexican Sweet) was dissected by limited proteolysis with subtilisin. Tubulin in taxol-stabilized microtubules was cleaved in a subtilisin concentration- and time-dependent manner. Immunoblotting of microtubules with antibodies having mapped epitopes on α- and β-tubulins revealed that cleavage initially removed ≤15 residues from the β-tubulin carboxyl terminus to produce αβ_s-microtubules. Subsequent cleavage occurred at an extreme site and an internal site within the α-tubulin carboxyl terminus. Electron microscopy revealed that αβ_s-microtubules were ultra structurally indistinguishable from uncleaved control αβ-micro-tubules. Quantitative polymer sedimentation showed that low temperature treatment (0°C) caused significant depolymerization of αβ-microtubules, but little depolymerization of αβ_s-microtubules. Ca²⁺ enhanced the cold-induced depolymerization of both αβ- and αβ_s-microtubules. However, αβ_s-microtubules were significantly more stable to depolymerization by cold and Ca²⁺ than were αβ-micro-tubules. The results showed that maize microtubules containing shortened β-tubulin carboxyl termini are relatively resistant to the combined depolymerizing effects of cold and Ca²⁺. Thus, the extreme carboxyl terminus of β-tubulin is a crucial element of the plant tubulin regulatory domain and may be involved in the modulation of microtubule stability during the chilling response in plants.     

On and Around Microtubules: An Overview

Microtubules are hollow tubes some 25 nm in diameter participating in the eukaryotic cytoskeleton. They are built from αβ-tubulin heterodimers that associate to form protofilaments running lengthwise along the microtubule wall with the β-tubulin subunit facing the microtubule plus end conferring a structural polarity. The α- and β-tubulins are highly conserved. A third member of the tubulin family, γ-tubulin, plays a role in microtubule nucleation and assembly. Other members of the tubulin family appear to be involved in microtubule nucleation. Microtubule assembly is accompanied by hydrolysis of GTP associated with β-tubulin so that microtubules consist principally of ‘GDP-tubulin’ stabilized at the plus end by a short ‘cap’. An important property of microtubules is dynamic instability characterized by growth randomly interrupted by pauses and shrinkage. Many proteins interact with microtubules within the cell and are involved in essential functions such as microtubule growth, stabilization, destabilization, and interactions with chromosomes during cell division. The motor proteins kinesin and dynein use microtubules as pathways for transport and are also involved in cell division. Crystallography and electron microscopy are providing a structural basis for understanding the interactions of microtubules with antimitotic drugs, with motor proteins and with plus end tracking proteins.     

Microtubules in bacteria: Ancient tubulins build a five-protofilament homolog of the eukaryotic cytoskeleton

Microtubules play crucial roles in cytokinesis, transport, and motility, and are therefore superb targets for anti-cancer drugs. All tubulins evolved from a common ancestor they share with the distantly related bacterial cell division protein FtsZ, but while eukaryotic tubulins evolved into highly conserved microtubule-forming heterodimers, bacterial FtsZ presumably continued to function as single homopolymeric protofilaments as it does today. Microtubules have not previously been found in bacteria, and we lack insight into their evolution from the tubulin/FtsZ ancestor. Using electron cryomicroscopy, here we show that the tubulin homologs BtubA and BtubB form microtubules in bacteria and suggest these be referred to as "bacterial microtubules" (bMTs). bMTs share important features with their eukaryotic counterparts, such as straight protofilaments and similar protofilament interactions. bMTs are composed of only five protofilaments, however, instead of the 13 typical in eukaryotes. These and other results suggest that rather than being derived from modern eukaryotic tubulin, BtubA and BtubB arose from early tubulin intermediates that formed small microtubules. Since we show that bacterial microtubules can be produced in abundance in vitro without chaperones, they should be useful tools for tubulin research and drug screening.     

Delineating the interaction of combretastatin A-4 with αβ tubulin isotypes present in drug resistant human lung carcinoma using a molecular modeling approach

Abstract

Tubulin isotypes are known to regulate microtubule dynamic instability and contribute to the development of drug resistance in certain types of cancers. Combretastatin-A4 (CA-4) has a potent anti-mitotic, vascular disrupting and anti-angiogenic activity. It binds at the interface of αβ tubulin heterodimers and inhibits microtubules assembly. Interestingly, the CA-4 resistant human lung carcinoma shows alteration of βI and βIII isotype levels, a higher expression of βI tubulin isotype and a decreased expression of βIII tubulin isotypes has been reported in drug resistant cell lines. However, the origin of CA-4 resistance in lung carcinoma is not well understood. Here, we investigate the interaction and binding affinities of αβI, αβIIb, αβIII and αβIVa tubulin isotypes with CA-4, employing molecular modeling approaches. Sequence analysis shows that variations in residue composition at the CA-4 binding pocket of βI, βIII and βIVa tubulin isotypes when compared to template βIIb isotype. Molecular docking result shows that the CA-4 prefers ‘cis’ conformation in all αβ-tubulin isotypes. Molecular dynamics simulation reveal role of H7 helix, T7 loop and H8 helix of β-tubulin in lower binding affinity of αβI and αβIII isotypes for CA-4. The order of binding energy for CA-4 is αβIIb?>?αβIVa?>?αβI?>?αβIII. This suggest that drug resistance is induced in human lung carcinoma cells by altering the expression of β-tubulin isotypes namely βI and βIII which show lowest binding affinities. Our present study can help in designing potential CA-4 analogs against drug-resistant cancer cells showing altered expression of tubulin isotypes. Abbreviations: CA-4 combretastatin-A4

MD molecular dynamics

RMSD root mean square deviation

DSSP dictionary of secondary structure of proteins

VMD visual molecular dynamics

Communicated by Ramaswamy H. Sarma     

Comparative immunogold analysis of tubulin isoforms in the mouse sperm flagellum: Unique distribution of glutamylated tubulin

The distribution of different tubulin isoforms in the mouse sperm flagellum was studied using four site-directed antibodies to tubulin: DM1A and DM1B general anti α and β-tubulin, 6-11B-1 anti-acetylated α-tubulin, and GT335 anti-glutamylated α and β-tubulin. Quantitative immunogold analyses were performed on five regions of the flagellum: the middle piece, three successive regions of the principal piece, and the terminal piece. A uniform labeling was observed with DM1A and DM1B along the entire flagellum both for peripheral doublets and the central pair. Similar results were obtained with 6-11B-1 directed to acetylated α-tubulin, an N-terminal-modified tubulin isoform. In contrast, the labeling for glutamylated α and β-tubulin, C-terminal modified isoforms, was not uniform. The highest intensity was found in the middle piece and the terminal piece. The labeling which decreased significantly both for peripheral doublets and central pair along the principal piece was considered as a loss of glutamylated tubulin accessibility. From the middle piece to the end of the principal piece, this labeling was predominant in doublets 1-5-6, corresponding to the plane of the flagellar wave. However, the labeling for doublets 2-3-4-7-8-9 was heterogeneous, showing an increasing asymmetry. These results suggest that in the mammalian sperm cell model, the glutamylated tubulin might be involved in a functional heterogeneity among peripheral doublets of the flagellum. © 1996 Wiley-Liss, Inc.     

Enhancement of tubulin polymerization by Cl(-)-induced blockade of intrinsic GTPase

In growing neurite of neuronal cells, it is suggested that α/β-tubulin heterodimers assemble to form microtubule, and assembly of microtubule promotes neurite elongation. On the other hand, recent studies reveal importance of intracellular Cl(-) in regulation of various cellular functions such as cell cycle progression, differentiation, cell migration, and elongation of neurite in neuronal cells. In this study, we investigated effects of Cl(-) on in vitro tubulin polymerization. We found that efficiency of in vitro tubulin polymerization (the number of microtubule) was higher (3 to 5-fold) in Cl(-)-containing solutions than that in Cl(-)-free solutions containing Br(-) or NO(3)(-). On the other hand, GTPase activity of tubulin was lower (2/3-fold) in Cl(-)-containing solutions than that in Cl(-)-free solutions containing Br(-) or NO(3)(-). Efficiency of in vitro tubulin polymerization in solutions containing a non-hydrolyzable analogue of GTP (GpCpp) instead of GTP was much higher than that in the presence of GTP. Effects of replacement of GTP with GpCpp on in vitro tubulin polymerization was weaker in Cl(-) solutions (10-fold increases) than that in Br(-) or NO(3)(-) solutions (20-fold increases), although the efficiency of in vitro tubulin polymerization in Cl(-) solutions containing GpCpp was still higher than that in Br(-) or NO(3)(-) solutions containing GpCpp. Our results suggest that a part of stimulatory effects of Cl(-) on in vitro tubulin polymerization is mediated via an inhibitory effect on GTPase activity of tubulin, although Cl(-) would also regulate in vitro tubulin polymerization by factors other than an inhibitory effect on GTPase activity.     

原核生物微管蛋白家族研究进展

自从1992年确定细菌分裂的关键蛋白Fts Z属于微管蛋白家族以来,越来越多的细菌细胞骨架蛋白被发现。原核生物中的微管同源蛋白主要有Fts Z、Cet Z、Tub Z和Btub A/B等。它们与微管蛋白具有相似的三级结构,可以结合鸟嘌呤-5′-三磷酸(Guanosine triphosphate,GTP)自聚合成不同的线状原丝纤维结构:单线状原丝纤维、双螺旋纤维结构或聚集成束状结构,在细菌细胞分裂、维持细胞形态、质粒分离等诸多重要生理功能中起着重要作用。     

BtubA-BtubB Heterodimer Is an Essential Intermediate in Protofilament Assembly

Background

BtubA and BtubB are two tubulin-like genes found in the bacterium Prosthecobacter. Our work and a previous crystal structure suggest that BtubB corresponds to α−tubulin and BtubA to β−tubulin. A 1∶1 mixture of the two proteins assembles into tubulin-like protofilaments, which further aggregate into pairs and bundles. The proteins also form a BtubA/B heterodimer, which appears to be a repeating subunit in the protofilament.

Methodology/Principal Findings

We have designed point mutations to disrupt the longitudinal interfaces bonding subunits into protofilaments. The mutants are in two classes, within dimers and between dimers. We have characterized one mutant of each class for BtubA and BtubB. When mixed 1∶1 with a wild type partner, none of the mutants were capable of assembly. An excess of between-dimer mutants could depolymerize preformed wild type polymers, while within-dimer mutants had no activity.

Conclusions

An essential first step in assembly of BtubA + BtubB is formation of a heterodimer. An excess of between-dimer mutants depolymerize wild type BtubA/B by sequestering the partner wild type subunit into inactive dimers. Within-dimer mutants cannot form dimers and have no activity.     

In vitro formation of different tubulin polymers from purified tubulin of Ehrlich ascites tumor cells

Preparations of cycled tubulin from Ehrlich ascites tumor cells contain several acessory proteins; once or twice cycled microtubule preparations are usually composed of fibers 10 nm in diameter, but lack vimentin. Highly purified tubulin consists of α- and β-tubulin and a minor component which was identified by peptide mapping as a second β-chain. This pure tubulin is able to form in vitro at low concentrations (1 mg protein/ml) fibers of about 10 nm width, and at higher concentrations (3.5 mg protein/ml) normal microtubules.     

Structural mechanisms underlying nucleotide-dependent self-assembly of tubulin and its relatives

The alphabeta-tubulin dimer assembles into microtubules, essential polymers in all eukaryotic cells. Microtubules are highly dynamic, a property that derives from tubulin's GTPase activity. Both the bacterial homolog, FtsZ, and the recently discovered bacterial tubulins from Prosthecobacter self-assemble in a nucleotide-dependent manner into protofilaments similar to those that form the microtubule wall. A number of structural studies of alphabeta-tubulin, gamma-tubulin (the isoform involved in microtubule nucleation), FtsZ and bacterial tubulin, in a variety of nucleotide and polymerization states, have been reported in the past few years. These studies have revealed the similarities and differences between these structures and their possible functional implications. In particular, a two-state mechanism has been proposed for the recycling of alphabeta-tubulin during the microtubule disassembly-assembly cycle; this mechanism may be unique to eukaryotic dimeric tubulin and the microtubule structure.     

Evidence for a distinct ligand binding site on tubulin discovered through inhibition by GDP of paclitaxel-induced tubulin assembly in the absence of exogenous GTP

GDP inhibits paclitaxel-induced tubulin assembly without GTP when the tubulin bears GDP in the exchangeable site (E-site). Initially, we thought inhibition was mediated through the E-site, since small amounts of GTP or Mg²⁺, which favors GTP binding to the E-site, reduced inhibition by GDP. We thought trace GTP released from the nonexchangeable site (N-site) by tubulin denaturation was required for polymer nucleation, but microtubule length was unaffected by GDP. Further, enhancing polymer nucleation reduced inhibition by GDP. Other mechanisms involving the E-site were eliminated experimentally. Upon finding that ATP weakly inhibited paclitaxel-induced assembly, we concluded that another ligand binding site was responsible for these inhibitory effects, and we found that GDP was not binding at the taxoid, colchicine, or vinca sites. There may therefore be a lower affinity site on tubulin to which GDP can bind distinct from the E- and N-sites, possibly on α-tubulin, based on molecular modeling studies.     

Inhibition of tubulin self-assembly and tubulin-colchicine GTPase activity by guanosine 5'-(gamma-fluorotriphosphate)

The inhibitory effects of guanosine 5'-(gamma-fluorotriphosphate) [GTP(gamma F)] on both the polymerization and the colchicine-dependent GTPase activity of calf brain tubulin have been studied. The results demonstrate that this analogue of GTP, with a fluorine atom on the gamma-phosphate, is a reversible competitive dead-end inhibitor of the colchicine-induced GTPase activity with a K1 value of (1.8 +/- 0.6) X 10(-4) M. GTP(gamma F) did not promote assembly of tubulin from which the E-site guanine nucleotide had been removed. It binds to the exchangeable nucleotide site competitively with respect to GTP, diminishing both the rate and extent of tubulin polymerization. Treatment in terms of the Oosawa-Kasai model of the inhibitory effect of GTP(gamma F) on the assembly led to a value of Kdis = 1.1 X 10(-6) M for the complex GTP(gamma F)-tubulin. This analogue does not bind to the postulated third site. The growing of tubulin polymers at 37 degrees C was arrested by GTP(gamma F), and only limited depolymerization was induced by the addition of this analogue after assembly in the presence of GTP. This result confirms that the E-site is blocked in the polymer and that this analogue can bind only to the ends of the polymers. Sedimentation velocity and circular dichroism studies showed that the conformation of the tubulin-GTP(gamma F) complex is not identical with that of tubulin-GTP. This is caused by the replacement of the hydroxyl group in the gamma-phosphate by the fluorine group, which have 2.20- and 1.35-A van der Waals radii, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elucidation of the anticancer potential and tubulin isotype-specific interactions of β-sitosterol

Beta-sitosterol (β-SITO), a phytosterol present in many edible vegetables, has been reported to possess antineoplastic properties and cancer treatment potential. We have shown previously that it binds at a unique site (the ‘SITO-site’) compared to the colchicine binding site at the interface of α- and β-tubulin. In this study, we investigated the anticancer efficacy of β-SITO against invasive breast carcinoma using MCF-7 cells. Since ‘isotypes’ of β-tubulin show tissue-specific expression and many are associated with cancer drug resistance, using computer-assisted docking and atomistic molecular dynamic simulations, we also examined its binding interactions to all known isotypes of β-tubulin in αβ-tubulin dimer. β-SITO inhibited MCF-7 cell viability by up to 50%, compared to vehicle-treated control cells. Indicating its antimetastatic potential, the phytosterol strongly inhibited cell migration. Immunofluorescence imaging of β-SITO-treated MCF-7 cells exhibited disruption of the microtubules and chromosome organization. Far-UV circular dichroism spectra indicated loss of helical stability in tubulin when bound to β-SITO. Docking and MD simulation studies, combined with MM-PBSA and MM-GBSA calculations revealed that β-SITO preferentially binds with specific β-tubulin isotypes (β_II and β_III) in the αβ-tubulin dimer. Both these β-tubulin isotypes have been implicated in drug resistance against tubulin-targeted chemotherapeutics. Our data show the tubulin-targeted anticancer potential of β-SITO, and its potential clinical utility against β_II and β_III isotype-overexpressing neoplasms.     

A novel acetylation of β-tubulin by San modulates microtubule polymerization via down-regulating tubulin incorporation

Dynamic instability is a critical property of microtubules (MTs). By regulating the rate of tubulin polymerization and depolymerization, cells organize the MT cytoskeleton to accommodate their specific functions. Among many processes, posttranslational modifications of tubulin are implicated in regulating MT functions. Here we report a novel tubulin acetylation catalyzed by acetyltransferase San at lysine 252 (K252) of β-tubulin. This acetylation, which is also detected in vivo, is added to soluble tubulin heterodimers but not tubulins in MTs. The acetylation-mimicking K252A/Q mutants were incorporated into the MT cytoskeleton in HeLa cells without causing any obvious MT defect. However, after cold-induced catastrophe, MT regrowth is accelerated in San-siRNA cells while the incorporation of acetylation-mimicking mutant tubulins is severely impeded. K252 of β-tubulin localizes at the interface of α-/β-tubulins and interacts with the phosphate group of the α-tubulin-bound GTP. We propose that the acetylation slows down tubulin incorporation into MTs by neutralizing the positive charge on K252 and allowing tubulin heterodimers to adopt a conformation that disfavors tubulin incorporation.     

Structural model of a complex between the heterotrimeric G protein, Gsalpha, and tubulin

A number of studies have demonstrated interplay between the cytoskeleton and G protein signaling. Many of these studies have determined a specific interaction between tubulin, the building block of microtubules, and G proteins. The alpha subunits of some heterotrimeric G proteins, including Gsalpha, have been shown to interact strongly with tubulin. Binding of Galpha to tubulin results in increased dynamicity of microtubules due to activation of GTPase of tubulin. Tubulin also activates Gsalpha via a direct transfer of GTP between these molecules. Structural insight into the interaction between tubulin and Gsalpha was required, and was determined, in this report, through biochemical and molecular docking techniques. Solid phase peptide arrays suggested that a portion of the amino terminus, alpha2-beta4 (the region between switch II and switch III) and alpha3-beta5 (just distal to the switch III region) domains of Gsalpha are important for interaction with tubulin. Molecular docking studies revealed the best-fit models based on the biochemical data, showing an interface between the two molecules that includes the adenylyl cyclase/Gbetagamma interaction regions of Gsalpha and the exchangeable nucleotide-binding site of tubulin. These structural models explain the ability of tubulin to facilitate GTP exchange on Galpha and the ability of Galpha to activate tubulin GTPase.     

Site and timing of synthesis of tubulin and other proteins during oogenesis in Drosophila melanogaster

Protein synthetic patterns during oogenesis in Drosophila melanogaster were examined; in particular the site, time, and rate of tubulin synthesis and accumulation during oogenesis were determined. Ovarian proteins were labeled with [³⁵S]methionine in vivo or in organ culure in vitro, and the proteins synthesized in egg chambers of specific developmental stages displayed by two-dimensional gel electrophoresis. A dissection technique was devised to examine proteins synthesized in each of the three cell types present in stage 10B egg chambers. The majority of proteins which were resolved by two-dimensional gel electrophoresis, including tubulin and actin, were synthesized throughout oogenesis and, at least to some extent, in each of the stage 10B cell types. Protein synthesis specific to developmental stage and/or cell type was also observed; for example, two nonchorion proteins were synthesized only in follicle cells and primarily at stage 10. A sensitive and specific radioimmune assay was developed in order to quantitate tubulin accumulation. Synthesis of several α-tubulin subunits and one β-tubulin subunit was observed. The tubulin content per egg chamber increased from 3 ng in stage 9 to 17 ng in stage 14, a period of about 13 hr. An accumulation rate of 1 ng/hr suggests that tubulin mRNA can account for about 4% of the total, nonmitochondrial, poly(A)⁺ RNA of the egg. Analysis of separated cell types at stage 10B revealed that both the follicle and nurse cells synthesize and accumulate appreciable amounts of tubulin. The stage 10B oocyte contains relatively little tubulin but actively synthesizes it. These two complementary analyses demonstrate that the tubulin present in the egg is synthesized within the oocyte-nurse cell syncytium, first in the nurse cells and later in the oocyte.