c-Abl kinase-mediated phosphorylation of γ-tubulin promotes γ-tubulin ring complexes assembly and microtubule nucleation

Cytoskeletal microtubules (MTs) are nucleated from γ-tubulin ring complexes (γTuRCs) located at MT organizing centers (MTOCs), such as the centrosome. However, the exact regulatory mechanism of γTuRC assembly is not fully understood. Here, we showed that the nonreceptor tyrosine kinase c-Abl was associated with and phosphorylated γ-tubulin, the essential component of the γTuRC, mainly on the Y443 residue by in vivo (immunofluorescence and immunoprecipitation) or in vitro (surface plasmon resonance) detection. We further demonstrated that phosphorylation deficiency significantly impaired γTuRC assembly, centrosome construction, and MT nucleation. c-Abl/Arg deletion and γ-tubulin Y443F mutation resulted in an abnormal morphology and compromised spindle function during mitosis, eventually causing uneven chromosome segregation. Our findings reveal that γTuRC assembly and nucleation function are regulated by Abl kinase-mediated γ-tubulin phosphorylation, revealing a fundamental mechanism that contributes to the maintenance of MT function.     

Characterization of the Human Homologue of the Yeast Spc98p and Its Association with γ-Tubulin

A trimeric complex formed by Tub4p, the budding yeast γ-tubulin, and the two spindle pole body components, Spc98p and Spc97p, has recently been characterized in Saccharomyces cerevisiae. We reasoned that crucial functions, such as the control of microtubule nucleation, could be maintained among divergent species. SPC98-related sequences were searched in dbEST using the BLASTN program. Primers derived from the human expressed sequence tag matching SPC98 were used to clone the 5′ and 3′ cDNA ends by rapid amplification of cDNA ends (RACE)-PCR. The human Spc98 cDNA presents an alternative splicing at the 3′ end. The deduced protein possesses 22% identity and 45% similarity with the yeast homologue. We further report that the human Spc98p, like γ-tubulin, is concentrated at the centrosome, although a large fraction is found in cytosolic complexes. Sucrose gradient sedimentation of the cytosolic fraction and immunoprecipitation experiments demonstrate that both γ-tubulin and HsSpc98p are in the same complex. Interestingly, Xenopus sperm centrosomes, which are incompetent for microtubule nucleation before their activation in the egg cytoplasm, were found to contain similar amounts of both Spc98p and γ-tubulin to human somatic centrosomes, which are competent for microtubule nucleation. Finally, affinity-purified antibodies against Spc98p inhibit microtubule nucleation on isolated centrosomes, as well as in microinjected cells, suggesting that this novel protein is indeed required for the nucleation reaction.     

An extended γ-tubulin ring functions as a stable platform in microtubule nucleation

γ-Tubulin complexes are essential for microtubule (MT) nucleation. The γ-tubulin small complex (γ-TuSC) consists of two molecules of γ-tubulin and one molecule each of Spc97 and Spc98. In vitro, γ-TuSCs oligomerize into spirals of 13 γ-tubulin molecules per turn. However, the properties and numbers of γ-TuSCs at MT nucleation sites in vivo are unclear. In this paper, we show by fluorescence recovery after photobleaching analysis that γ-tubulin was stably integrated into MT nucleation sites and was further stabilized by tubulin binding. Importantly, tubulin showed a stronger interaction with the nucleation site than with the MT plus end, which probably provides the basis for MT nucleation. Quantitative analysis of γ-TuSCs on single MT minus ends argued for nucleation sites consisting of approximately seven γ-TuSCs with approximately three additional γ-tubulin molecules. Nucleation and anchoring of MTs required the same number of γ-tubulin molecules. We suggest that a spiral of seven γ-TuSCs with a slight surplus of γ-tubulin nucleates MTs in vivo.     

Spc98p Directs the Yeast γ-Tubulin Complex into the Nucleus and Is Subject to Cell Cycle-dependent Phosphorylation on the Nuclear Side of the Spindle Pole Body

In the yeast Saccharomyces cerevisiae, microtubules are organized by the spindle pole body (SPB), which is embedded in the nuclear envelope. Microtubule organization requires the γ-tubulin complex containing the γ-tubulin Tub4p, Spc98p, and Spc97p. The Tub4p complex is associated with cytoplasmic and nuclear substructures of the SPB, which organize the cytoplasmic and nuclear microtubules. Here we present evidence that the Tub4p complex assembles in the cytoplasm and then either binds to the cytoplasmic side of the SPB or is imported into the nucleus followed by binding to the nuclear side of the SPB. Nuclear import of the Tub4p complex is mediated by the essential nuclear localization sequence of Spc98p. Our studies also indicate that Spc98p in the Tub4p complex is phosphorylated at the nuclear, but not at the cytoplasmic, side of the SPB. This phosphorylation is cell cycle dependent and occurs after SPB duplication and nucleation of microtubules by the new SPB and therefore may have a role in mitotic spindle function. In addition, activation of the mitotic checkpoint stimulates Spc98p phosphorylation. The kinase Mps1p, which functions in SPB duplication and mitotic checkpoint control, seems to be involved in Spc98p phosphorylation. Our results also suggest that the nuclear and cytoplasmic Tub4p complexes are regulated differently.     

Multicolor BiFC analysis of competition among G protein β and γ subunit interactions

We have applied multicolor BiFC to study the association preferences of G protein β and γ subunits in living cells. Cells co-express multiple isoforms of β and γ subunits, most of which can form complexes. Although many βγ complexes exhibit similar properties when assayed in reconstituted systems, knockout experiments in vivo suggest that individual isoforms have unique functions. BiFC makes it possible to correlate βγ complex formation with functionality in intact cells by comparing the amounts of fluorescent βγ complexes with their abilities to modulate effector proteins. The relative predominance of specific βγ complexes in vivo is not known. To address this issue, multicolor BiFC can determine the association preferences of β and γ subunits by simultaneously visualizing the two fluorescent complexes formed when β or γ subunits fused to amino terminal fragments of yellow fluorescent protein (YFP-N) and cyan fluorescent protein (CFP-N) compete to interact with limiting amounts of a common γ or β subunit, respectively, fused to a carboxyl terminal fragment of CFP (CFP-C). Multicolor BiFC also makes it possible to determine the roles of interacting proteins in the subcellular targeting of complexes, study the formation of protein complexes that are unstable under isolation conditions, determine the roles of co-expressed proteins in regulating the association preferences of interacting proteins, and visualize dynamic events affecting multiple protein complexes. These approaches can be applied to studying the assembly and functions of a wide variety of protein complexes in the context of a living cell.     

The Mammalian γ-Tubulin Complex Contains Homologues of the Yeast Spindle Pole Body Components Spc97p and Spc98p

γ-Tubulin is a universal component of microtubule organizing centers where it is believed to play an important role in the nucleation of microtubule polymerization. γ-Tubulin also exists as part of a cytoplasmic complex whose size and complexity varies in different organisms. To investigate the composition of the cytoplasmic γ-tubulin complex in mammalian cells, cell lines stably expressing epitope-tagged versions of human γ-tubulin were made. The epitope-tagged γ-tubulins expressed in these cells localize to the centrosome and are incorporated into the cytoplasmic γ-tubulin complex. Immunoprecipitation of this complex identifies at least seven proteins, with calculated molecular weights of 48, 71, 76, 100, 101, 128, and 211 kD. We have identified the 100- and 101-kD components of the γ-tubulin complex as homologues of the yeast spindle pole body proteins Spc97p and Spc98p, and named the corresponding human proteins hGCP2 and hGCP3. Sequence analysis revealed that these proteins are not only related to their respective homologues, but are also related to each other. GCP2 and GCP3 colocalize with γ-tubulin at the centrosome, cosediment with γ-tubulin in sucrose gradients, and coimmunoprecipitate with γ-tubulin, indicating that they are part of the γ-tubulin complex. The conservation of a complex involving γ-tubulin, GCP2, and GCP3 from yeast to mammals suggests that structurally diverse microtubule organizing centers such as the yeast spindle pole body and the animal centrosome share a common molecular mechanism for microtubule nucleation.     

A Mutational Analysis Identifies Three Functional Regions of the Spindle Pole Component Spc110p in Saccharomyces cerevisiae

The central coiled coil of the essential spindle pole component Spc110p spans the distance between the central and inner plaques of the Saccharomyces cerevisiae spindle pole body (SPB). The carboxy terminus of Spc110p, which binds calmodulin, resides at the central plaque, and the amino terminus resides at the inner plaque from which nuclear microtubules originate. To dissect the functions of Spc110p, we created temperature-sensitive mutations in the amino and carboxy termini. Analysis of the temperature-sensitive spc110 mutations and intragenic complementation analysis of the spc110 alleles defined three functional regions of Spc110p. Region I is located at the amino terminus. Region II is located at the carboxy-terminal end of the coiled coil, and region III is the previously defined calmodulin-binding site. Overexpression of SPC98 suppresses the temperature sensitivity conferred by mutations in region I but not the phenotypes conferred by mutations in the other two regions, suggesting that the amino terminus of Spc110p is involved in an interaction with the γ-tubulin complex composed of Spc97p, Spc98p, and Tub4p. Mutations in region II lead to loss of SPB integrity during mitosis, suggesting that this region is required for the stable attachment of Spc110p to the central plaque. Our results strongly argue that Spc110p links the γ-tubulin complex to the central plaque of the SPB.     

p87 and p101 Subunits Are Distinct Regulators Determining Class IB Phosphoinositide 3-Kinase (PI3K) Specificity

Class I_B phosphoinositide 3-kinase γ (PI3Kγ) comprises a single catalytic p110γ subunit, which binds to two non-catalytic subunits, p87 or p101, and controls a plethora of fundamental cellular responses. The non-catalytic subunits are assumed to be redundant adaptors for Gβγ enabling G-protein-coupled receptor-mediated regulation of PI3Kγ. Growing experimental data provide contradictory evidence. To elucidate the roles of the non-catalytic subunits in determining the specificity of PI3Kγ, we tested the impact of p87 and p101 in heterodimeric p87-p110γ and p101-p110γ complexes on the modulation of PI3Kγ activity in vitro and in living cells. RT-PCR, biochemical, and imaging data provide four lines of evidence: (i) specific expression patterns of p87 and p101, (ii) up-regulation of p101, providing the basis to consider p87 as a protein forming a constitutively and p101 as a protein forming an inducibly expressed PI3Kγ, (iii) differences in basal and stimulated enzymatic activities, and (iv) differences in complex stability, all indicating apparent diversity within class I_B PI3Kγ. In conclusion, expression and activities of PI3Kγ are modified differently by p87 and p101 in vitro and in living cells, arguing for specific regulatory roles of the non-catalytic subunits in the differentiation of PI3Kγ signaling pathways.     

Genome-wide haploinsufficiency screen reveals a novel role for γ-TuSC in spindle organization and genome stability

How subunit dosage contributes to the assembly and function of multimeric complexes is an important question with implications in understanding biochemical, evolutionary, and disease mechanisms. Toward identifying pathways that are susceptible to decreased gene dosage, we performed a genome-wide screen for haploinsufficient (HI) genes that guard against genome instability in Saccharomyces cerevisiae. This led to the identification of all three genes (SPC97, SPC98, and TUB4) encoding the evolutionarily conserved γ-tubulin small complex (γ-TuSC), which nucleates microtubule assembly. We found that hemizygous γ-TuSC mutants exhibit higher rates of chromosome loss and increases in anaphase spindle length and elongation velocities. Fluorescence microscopy, fluorescence recovery after photobleaching, electron tomography, and model convolution simulation of spc98/+ mutants revealed improper regulation of interpolar (iMT) and kinetochore (kMT) microtubules in anaphase. The underlying cause is likely due to reduced levels of Tub4, as overexpression of TUB4 suppressed the spindle and chromosome segregation defects in spc98/+ mutants. We propose that γ-TuSC is crucial for balanced assembly between iMTs and kMTs for spindle organization and accurate chromosome segregation. Taken together, the results show how gene dosage studies provide critical insights into the assembly and function of multisubunit complexes that may not be revealed by using traditional studies with haploid gene deletion or conditional alleles.     

A Genetic Analysis of Interactions with Spc110p Reveals Distinct Functions of Spc97p and Spc98p, Components of the Yeast γ-Tubulin Complex

The spindle pole body (SPB) in Saccharomyces cerevisiae functions as the microtubule-organizing center. Spc110p is an essential structural component of the SPB and spans between the central and inner plaques of this multilamellar organelle. The amino terminus of Spc110p faces the inner plaque, the substructure from which spindle microtubules radiate. We have undertaken a synthetic lethal screen to identify mutations that enhance the phenotype of the temperature-sensitive spc110–221 allele, which encodes mutations in the amino terminus. The screen identified mutations in SPC97 and SPC98, two genes encoding components of the Tub4p complex in yeast. The spc98–63 allele is synthetic lethal only with spc110 alleles that encode mutations in the N terminus of Spc110p. In contrast, the spc97 alleles are synthetic lethal with spc110 alleles that encode mutations in either the N terminus or the C terminus. Using the two-hybrid assay, we show that the interactions of Spc110p with Spc97p and Spc98p are not equivalent. The N terminus of Spc110p displays a robust interaction with Spc98p in two different two-hybrid assays, while the interaction between Spc97p and Spc110p is not detectable in one strain and gives a weak signal in the other. Extra copies of SPC98 enhance the interaction between Spc97p and Spc110p, while extra copies of SPC97 interfere with the interaction between Spc98p and Spc110p. By testing the interactions between mutant proteins, we show that the lethal phenotype in spc98–63 spc110–221 cells is caused by the failure of Spc98–63p to interact with Spc110–221p. In contrast, the lethal phenotype in spc97–62 spc110–221 cells can be attributed to a decreased interaction between Spc97–62p and Spc98p. Together, these studies provide evidence that Spc110p directly links the Tub4p complex to the SPB. Moreover, an interaction between Spc98p and the amino-terminal region of Spc110p is a critical component of the linkage, whereas the interaction between Spc97p and Spc110p is dependent on Spc98p.     

MT1-MMP Initiates Activation of pro-MMP-2 and Integrin αvβ3 Promotes Maturation of MMP-2 in Breast Carcinoma Cells

We evaluated cellular mechanisms involved in the activation pathway of matrix prometalloproteinase-2 (pro-MMP-2), an enzyme implicated in the malignant progression of many tumor types. Membrane type-1 matrix metalloproteinase (MT1-MMP) cleaves the N-terminal prodomain of pro-MMP-2 thus generating the activation intermediate that then matures into the fully active enzyme of MMP-2. Our results provide evidence on how a collaboration between MT1-MMP and integrin αvβ3 promotes more efficient activation and specific, transient docking of the activation intermediate and, further, the mature, active enzyme of MMP-2 at discrete regions of cells. We show that coexpression of MT1-MMP and integrin αvβ3 in MCF7 breast carcinoma cells specifically enhances in trans autocatalytic maturation of MMP-2. The association of MMP-2′s C-terminal hemopexin-like domain with those molecules of integrin αvβ3 which are proximal to MT1-MMP facilitates MMP-2 maturation. Vitronectin, a specific ligand of integrin αvβ3, competitively blocked the integrin-dependent maturation of MMP-2. Immunofluorescence and immunoprecipitation studies supported clustering of MT1-MMP and integrin αvβ3 at discrete regions of the cell surface. Evidently, the identified mechanisms appear to be instrumental to clustering active MMP-2 directly at the invadopodia and invasive front of αvβ3-expressing cells or in their close vicinity, thereby accelerating tumor cell locomotion.     

Synthesis, biological evaluation and molecular modelling of sulfonohydrazides as selective PI3K p110alpha inhibitors

A series of 2-methyl-5-nitrobenzenesulfonohydrazides were prepared and evaluated as inhibitors of PI3K. An isoquinoline derivative shows good selectivity for the p110α isoform over p110β and p110δ, and also demonstrates good in vitro activity in a cell proliferation assay. Molecular modelling provides a rationalisation for the observed SAR.     

TACC3 Protein Regulates Microtubule Nucleation by Affecting γ-Tubulin Ring Complexes

Centrosome-mediated microtubule nucleation is essential for spindle assembly during mitosis. Although γ-tubulin complexes have primarily been implicated in the nucleation process, details of the underlying mechanisms remain poorly understood. Here, we demonstrated that a member of the human transforming acidic coiled-coil (TACC) protein family, TACC3, plays a critical role in microtubule nucleation at the centrosome. In mitotic cells, TACC3 knockdown substantially affected the assembly of microtubules in the astral region and impaired microtubule nucleation at the centrosomes. The TACC3 depletion-induced mitotic phenotype was rescued by expression of the TACC3 C terminus predominantly consisting of the TACC domain, suggesting that the TACC domain plays an important role in microtubule assembly. Consistently, experiments with the recombinant TACC domain of TACC3 demonstrated that this domain possesses intrinsic microtubule nucleating activity. Co-immunoprecipitation and sedimentation experiments revealed that TACC3 mediates interactions with proteins of both the γ-tubulin ring complex (γ-TuRC) and the γ-tubulin small complex (γ-TuSC). Interestingly, TACC3 depletion resulted in reduced levels of γ-TuRC and increased levels of γ-TuSC, indicating that the assembly of γ-TuRC from γ-TuSC requires TACC3. Detailed analyses suggested that TACC3 facilitates the association of γ-TuSC-specific proteins with the proteins known to be involved in the assembly of γ-TuRC. Consistent with such a role for TACC3, the suppression of TACC3 disrupted localization of γ-TuRC proteins to the centrosome. Our findings reveal that TACC3 is involved in the regulation of microtubule nucleation at the centrosome and functions in the stabilization of the γ-tubulin ring complex assembly.     

γ-Tubulin ring complexes regulate microtubule plus end dynamics

γ-Tubulin is critical for the initiation and regulation of microtubule (MT) assembly. In Drosophila melanogaster, it acts within two main complexes: the γ-tubulin small complex (γ-TuSC) and the γ-tubulin ring complex (γ-TuRC). Proteins specific of the γ-TuRC, although nonessential for viability, are required for efficient mitotic progression. Until now, their role during interphase remained poorly understood. Using RNA interference in Drosophila S2 cells, we show that the γ-TuRC is not critical for overall MT organization. However, depletion of any component of this complex results in an increase of MT dynamics. Combined immunofluorescence and live imaging analysis allows us to reveal that the γ-TuRC localizes along interphase MTs and that distal γ-tubulin spots match with sites of pause or rescue events. We propose that, in addition to its role in nucleation, the γ-TuRC associated to MTs may regulate their dynamics by limiting catastrophes.     

Reconstitution of the recombinant human γ-tubulin ring complex

Cryo-electron microscopy recently resolved the structure of the vertebrate γ-tubulin ring complex (γ-TuRC) purified from Xenopus laevis egg extract and human cells to near-atomic resolution. These studies clarified the arrangement and stoichiometry of γ-TuRC components and revealed that one molecule of actin and the small protein MZT1 are embedded into the complex. Based on this structural census of γ-TuRC core components, we developed a recombinant expression system for the reconstitution and purification of human γ-TuRC from insect cells. The recombinant γ-TuRC recapitulates the structure of purified native γ-TuRC and has similar functional properties in terms of microtubule nucleation and minus end capping. This recombinant system is a central step towards deciphering the activation mechanisms of the γ-TuRC and the function of individual γ-TuRC core components.     

Characterisation of the beta-tubulin gene of Cylicocyclus nassatus

mRNA and genomic DNA were isolated from adult Cylicocyclus nassatus, and the mRNA was reverse transcribed. The cDNA was PCR amplified using degenerate primers designed according to the alignment of the β-tubulin amino acid sequences of other species. To complete the coding sequence, the 3′ end was amplified with the 3′-RACE, and for amplification of the 5′ end the SL1-primer was used. The cDNA of the β-tubulin gene of C. nassatus spans 1429 bp and encodes a protein of 448 amino acids. Specific primers were developed from the cDNA sequence to amplify the genomic DNA sequence and to analyse the genomic organisation of the β-tubulin gene. The complete sequence of the genomic DNA of the β-tubulin gene of C. nassatus has a size of 2652 bp and is organised into nine exons and eight introns. The identities with the exons of the gru-1 β-tubulin gene of Haemonchus contortus range between 79% and 97%.     

Loss of α-Tubulin Acetylation Is Associated with TGF-β-induced Epithelial-Mesenchymal Transition

The epithelial-to-mesenchymal transition (EMT) is a process by which differentiated epithelial cells reprogram gene expression, lose their junctions and polarity, reorganize their cytoskeleton, increase cell motility and assume a mesenchymal morphology. Despite the critical functions of the microtubule (MT) in cytoskeletal organization, how it participates in EMT induction and maintenance remains poorly understood. Here we report that acetylated α-tubulin, which plays an important role in microtubule (MT) stabilization and cell morphology, can serve as a novel regulator and marker of EMT. A high level of acetylated α-tubulin was correlated with epithelial morphology and it profoundly decreased during TGF-β-induced EMT. We found that TGF-β increased the activity of HDAC6, a major deacetylase of α-tubulin, without affecting its expression levels. Treatment with HDAC6 inhibitor tubacin or TGF-β type I receptor inhibitor SB431542 restored the level of acetylated α-tubulin and consequently blocked EMT. Our results demonstrate that acetylated α-tubulin can serve as a marker of EMT and that HDAC6 represents an important regulator during EMT process.     

NMR spectroscopy of α-crystallin. Insights into the structure, interactions and chaperone action of small heat-shock proteins

The subunit molecular mass of α-crystallin, like many small heat-shock proteins (sHsps), is around 20 kDa although the protein exists as a large aggregate of average mass around 800 kDa. Despite this large size, a well-resolved ¹H NMR spectrum is observed for α-crystallin which arises from short, polar, highly-flexible and solvent-exposed C-terminal extensions in each of the subunits, αA- and αB-crystallin. These extensions are not involved in interactions with other proteins (e.g. β- and γ-crystallins) under non-chaperone conditions. As determined by NMR studies on mutants of αA-crystallin with alterations in its C-terminal extension, the extensions have an important role in acting as solubilising agents for the relatively-hydrophobic α-crystallin molecule and the high-molecular-weight (HMW) complex that forms during the chaperone action. The related sHsp, Hsp25, also exhibits a flexible C-terminal extension. Under chaperone conditions, and in the HMW complex isolated from old lenses, the C-terminal extension of the αA-crystallin subunit maintains its flexibility whereas the αB-crystallin subunit loses, at least partially, its flexibility, implying that it is involved in interaction with the ‘substrate’ protein. The conformation of ‘substrate’ proteins when they interact with α-crystallin has been probed by ¹H NMR spectroscopy and it is concluded that α-crystallin interacts with ‘substrate’ proteins that are in a disordered molten globule state, but only when this state is on its way to large-scale aggregation and precipitation. By monitoring the ¹H and ³¹P NMR spectra of α-crystallin in the presence of increasing concentations of urea, it is proposed that α-crystallin adopts a two-domain structure with the larger C-terminal domain unfolding first in the presence of denaturant. All these data have been combined into a model for the quaternary structure of α-crystallin. The model has two layers each of approximately 40 subunits arranged in an annulus or toroid. A large central cavity is present whose entrance is ringed by the flexible C-terminal extensions. A large hydrophobic region in the aggregate is exposed to solution and is available for interaction with ‘substrate’ proteins during the chaperone action.     

Functional expression and sites of gene transcription of a novel α subunit of the GABAA receptor in rat brain

Two α subunits of the gabaa receptor in rat brain have been identified by molecular cloning. The deduced polypeptide sequences share major characteristics with other chemically gated ion channel proteins. One polypeptide represents the rat homologue of the α3 subunit previously cloned from bovine brain [14], while the other polypeptide is a yet unknown subunit, termed α5. When coexpressed with the β1 subunit in Xenopus oocytes the receptors containing the α5 subunit revealed a higher sensitivity to GABA than receptors expressed from α1 + β1 subunits or α3 + β1 subunits (K_a = 1 μM, 13 μM and 14 μM, respectively). The α5 subunit was expressed only in a few brain areas such as cerebral cortex, hippocampal formation and olfactory bulb granular layer as shown by in situ hybridization histochemistry. Since the mRNA of the α5 subunit was colocalized with the αl and α3 subunits only in cerebral cortex and in the hippocampal formation the α5 subunit may be part of distinct GABA_A receptors in neuronal populations within the olfactory bulb.