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The γ-tubulin ring complex (γ-TuRC) is a structural template for de novo microtubule assembly from α/β-tubulin units. The isolated vertebrate γ-TuRC assumes an asymmetric, open structure deviating from microtubule geometry, suggesting that γ-TuRC closure may underlie regulation of microtubule nucleation. Here, we isolate native γ-TuRC-capped microtubules from Xenopus laevis egg extract nucleated through the RanGTP-induced pathway for spindle assembly and determine their cryo-EM structure. Intriguingly, the microtubule minus end-bound γ-TuRC is only partially closed and consequently, the emanating microtubule is locally misaligned with the γ-TuRC and asymmetric. In the partially closed conformation of the γ-TuRC, the actin-containing lumenal bridge is locally destabilised, suggesting lumenal bridge modulation in microtubule nucleation. The microtubule-binding protein CAMSAP2 specifically binds the minus end of γ-TuRC-capped microtubules, indicating that the asymmetric minus end structure may underlie recruitment of microtubule-modulating factors for γ-TuRC release. Collectively, we reveal a surprisingly asymmetric microtubule minus end protofilament organisation diverging from the regular microtubule structure, with direct implications for the kinetics and regulation of nucleation and subsequent modulation of microtubules during spindle assembly. Synopsis: The universal microtubule nucleator γ-TuRC assumes a microtubule-incompatible open conformation prior to nucleation. This work finds it transitioning to a partially closed conformation, with local misalignments to the nucleated microtubule in the native RanGTP-dependent nucleation pathway. Cryo-EM analysis describes the structure of the γ-TuRC-capped microtubule minus end natively nucleated through the RanGTP pathway in Xenopus laevis egg extracts. The γ-TuRC conformation approaches but does not reach microtubule lattice symmetry after microtubule nucleation. The associated microtubule minus end is asymmetric and some of its protofilaments are misaligned with the partially closed γ-TuRC. The architecture of the γ-TuRC and the attached microtubule facilitates selective binding of microtubule-modulating protein CAMSAP2 at the γ-TuRC-capped microtubule minus end. Cryo-EM analysis of native γ-TuRC-capped microtubules from frog egg extracts shows a transition from an open to a partly closed conformation with local misalignments between nucleator and nucleated microtubule. [ABSTRACT FROM AUTHOR]

γ-Tubulin typically forms a ring-shaped complex with 5 related γ-tubulin complex proteins (GCP2 to GCP6), and this γ-tubulin ring complex (γTuRC) serves as a template for microtubule (MT) nucleation in plants and animals. While the γTuRC takes part in MT nucleation in most eukaryotes, in fungi such events take place robustly with just the γ-tubulin small complex (γTuSC) assembled by γ-tubulin plus GCP2 and GCP3. To explore whether the γTuRC is the sole functional γ-tubulin complex in plants, we generated 2 mutants of the GCP6 gene encoding the largest subunit of the γTuRC in Arabidopsis thaliana. Both mutants showed similar phenotypes of dwarfed vegetative growth and reduced fertility. The gcp6 mutant assembled the γTuSC, while the wild-type cells had GCP6 join other GCPs to produce the γTuRC. Although the gcp6 cells had greatly diminished γ-tubulin localization on spindle MTs, the protein was still detected there. The gcp6 cells formed spindles that lacked MT convergence and discernable poles; however, they managed to cope with the challenge of MT disorganization and were able to complete mitosis and cytokinesis. Our results reveal that the γTuRC is not the only functional form of the γ-tubulin complex for MT nucleation in plant cells, and that γ-tubulin-dependent, but γTuRC-independent, mechanisms meet the basal need of MT nucleation. Moreover, we show that the γTuRC function is more critical for the assembly of spindle MT array than for the phragmoplast. Thus, our findings provide insight into acentrosomal MT nucleation and organization.

Microtubules are protein cylinders with functions in cell motility, signal sensing, cell organization, intracellular transport, and chromosome segregation. One of the key properties of microtubules is their dynamic architecture, allowing them to grow and shrink in length by adding or removing copies of their basic subunit, the heterodimer αβ‐tubulin. In higher eukaryotes, de novo assembly of microtubules from αβ‐tubulin is initiated by a 2 MDa multi‐subunit complex, the gamma‐tubulin ring complex (γ‐TuRC). For many years, the structure of the γ‐TuRC and the function of its subunits remained enigmatic, although structural data from the much simpler yeast counterpart, the γ‐tubulin small complex (γ‐TuSC), were available. Two recent breakthroughs in the field, high‐resolution structural analysis and recombinant reconstitution of the complex, have revolutionized our knowledge about the architecture and function of the γ‐TuRC and will form the basis for addressing outstanding questions about biogenesis and regulation of this essential microtubule organizer. [ABSTRACT FROM AUTHOR]

Asymmetric astral microtubule organization drives the polarized orientation of the S. cerevisiae mitotic spindle and primes the invariant inheritance of the old spindle pole body (SPB, the yeast centrosome) by the bud. This model has anticipated analogous centrosome asymmetries featured in self-renewing stem cell divisions. We previously implicated Spc72, the cytoplasmic receptor for the gamma-tubulin nucleation complex, as the most upstream determinant linking SPB age, functional asymmetry and fate. Here we used structured illumination microscopy and biochemical analysis to explore the asymmetric landscape of nucleation sites inherently built into the spindle pathway and under the control of cyclin-dependent kinase (CDK). We show that CDK enforces Spc72 asymmetric docking by phosphorylating Nud1/centriolin. Furthermore, CDK-imposed order in the construction of the new SPB promotes the correct balance of nucleation sites between the nuclear and cytoplasmic faces of the SPB. Together these contributions by CDK inherently link correct SPB morphogenesis, age and fate.

Determining how microtubules (MTs) are nucleated is essential for understanding how the cytoskeleton assembles. While the MT nucleator, γ-tubulin ring complex (γ-TuRC) has been identified, precisely how γ-TuRC nucleates a MT remains poorly understood. Here, we developed a single molecule assay to directly visualize nucleation of a MT from purified Xenopus laevis γ-TuRC. We reveal a high γ-/αβ-tubulin affinity, which facilitates assembly of a MT from γ-TuRC. Whereas spontaneous nucleation requires assembly of 8 αβ-tubulins, nucleation from γ-TuRC occurs efficiently with a cooperativity of 4 αβ-tubulin dimers. This is distinct from pre-assembled MT seeds, where a single dimer is sufficient to initiate growth. A computational model predicts our kinetic measurements and reveals the rate-limiting transition where laterally associated αβ-tubulins drive γ-TuRC into a closed conformation. NME7, TPX2, and the putative activation domain of CDK5RAP2 do not enhance γ-TuRC-mediated nucleation, while XMAP215 drastically increases the nucleation efficiency by strengthening the longitudinal γ-/αβ-tubulin interaction.

Higher plants represent a large group of eukaryotes where centrosomes are absent. The functions of γ-tubulin small complexes (γ-TuSCs) and γ-tubulin ring complexes (γ-TuRCs) in metazoans and fungi in microtubule nucleation are well established and the majority of components found in the complexes are present in plants. However, plant microtubules are also nucleated in a γ-tubulin-dependent but γ-TuRC-independent manner. There is growing evidence that γ-tubulin is a microtubule nucleator without being complexed in γ-TuRC. Fibrillar arrays of γ-tubulin were demonstrated in plant and animal cells and the ability of γ-tubulin to assemble into linear oligomers/polymers was confirmed in vitro for both native and recombinant γ-tubulin. The functions of γ-tubulin as a template for microtubule nucleation or in promoting spontaneous nucleation is outlined. Higher plants represent an excellent model for studies on the role of γ-tubulin in nucleation due to their acentrosomal nature and high abundancy and conservation of γ-tubulin including its intrinsic ability to assemble filaments. The defining scaffolding or sequestration functions of plant γ-tubulin in microtubule organization or in nuclear processes will help our understanding of its cellular roles in eukaryotes.

Non‐erythroid spectrin or fodrin is present as part of the γ‐tubulin ring complex (γ‐TuRC) in brain tissue and brain derived cells. Here, we show that fodrin, which is otherwise known for providing structural support to the cell membrane, interacts directly with γ‐tubulin within the γ‐TuRC through a GRIP2‐like motif. Turbidometric analysis of microtubule polymerization with nucleation‐potent γ‐TuRC isolated from HEK‐293 cells that lack fodrin and the γ‐TuRC from goat brain that contains fodrin shows inefficiency of the latter to promote nucleation. The involvement of fodrin was confirmed by the reduction in the microtubule polymerization efficiency of HEK‐293 derived γ‐TuRCs upon addition of purified brain fodrin. Thus, the interaction of fodrin with gamma‐tubulin is responsible for its inhibitory effect on γ‐tubulin mediated microtubule nucleation. [ABSTRACT FROM AUTHOR]

Extracting isolated Drosophila centrosomes with 2 M KI generates salt-resistant scaffolds that lack the centrosomal proteins CP190, CP60, centrosomin, and gamma-tubulin. To clarify the role of these proteins in microtubule nucleation by centrosomes and to identify additional centrosome components required for nucleation, we have developed an in vitro complementation assay for centrosome function. Centrosome aster formation is reconstituted when these inactive, salt-stripped centrosome scaffolds are supplemented with a soluble fraction of a Drosophila embryo extract. The CP60 and CP190 can be removed from this extract without effect, whereas removing the gamma-tubulin destroys the complementing activity. Consistent with these results, we find no evidence that these three proteins form a complex together. Instead, gamma-tubulin is found in two distinct protein complexes of 240,000 and approximately 3,000,000 D. The larger complex, which is analogous to the Xenopus gamma-tubulin ring complex (gammaTuRC) (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578-583), is necessary but not sufficient for complementation. An additional factor found in the extract is required. These results provide the first evidence that the gammaTuRC is required for microtubule nucleation at the centrosome.

Extracting isolated Drosophila centrosomes with 2 M KI generates salt-resistant scaffolds that lack the centrosomal proteins CP190, CP60, centrosomin, and gamma-tubulin. To clarify the role of these proteins in microtubule nucleation by centrosomes and to identify additional centrosome components required for nucleation, we have developed an in vitro complementation assay for centrosome function. Centrosome aster formation is reconstituted when these inactive, salt-stripped centrosome scaffolds are supplemented with a soluble fraction of a Drosophila embryo extract. The CP60 and CP190 can be removed from this extract without effect, whereas removing the gamma-tubulin destroys the complementing activity. Consistent with these results, we find no evidence that these three proteins form a complex together. Instead, gamma-tubulin is found in two distinct protein complexes of 240,000 and approximately 3,000,000 D. The larger complex, which is analogous to the Xenopus gamma-tubulin ring complex (gammaTuRC) (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578-583), is necessary but not sufficient for complementation. An additional factor found in the extract is required. These results provide the first evidence that the gammaTuRC is required for microtubule nucleation at the centrosome.

Microtubules are major constituents of the cytoskeleton in all eukaryotic cells. They are essential for chromosome segregation during cell division, for directional intracellular transport and for building specialized cellular structures such as cilia or flagella. Their assembly has to be controlled spatially and temporally. For this, the cell uses multiprotein complexes containing γ-tubulin. γ-Tubulin has been found in two different types of complexes, γ-tubulin small complexes and γ-tubulin ring complexes. Binding to adaptors and activator proteins transforms these complexes into structural templates that drive the nucleation of new microtubules in a highly controlled manner. This review discusses recent advances on the mechanisms of assembly, recruitment and activation of γ-tubulin complexes at microtubule-organizing centres.

γ-Tubulin is a conserved member of the tubulin superfamily with a function in microtubule nucleation. Proteins of γ-tubulin complexes serve as nucleation templates as well as a majority of other proteins contributing to centrosomal and non-centrosomal nucleation, conserved across eukaryotes. There is a growing amount of evidence of γ-tubulin functions besides microtubule nucleation in transcription, DNA damage response, chromatin remodeling, and on its interactions with tumor suppressors. However, the molecular mechanisms are not well understood. Furthermore, interactions with lamin and SUN proteins of the LINC complex suggest the role of γ-tubulin in the coupling of nuclear organization with cytoskeletons. γ-Tubulin that belongs to the clade of eukaryotic tubulins shows characteristics of both prokaryotic and eukaryotic tubulins. Both human and plant γ-tubulins preserve the ability of prokaryotic tubulins to assemble filaments and higher-order fibrillar networks. γ-Tubulin filaments, with bundling and aggregating capacity, are suggested to perform complex scaffolding and sequestration functions. In this review, we discuss a plethora of γ-tubulin molecular interactions and cellular functions, as well as recent advances in understanding the molecular mechanisms behind them.

In textbooks, the mitotic spindles of plants are often described separately from those of animals. How do they differ at the molecular and mechanistic levels? In this chapter, we first outline the process of mitotic spindle assembly in animals and land plants. We next discuss the conservation of spindle assembly factors based on database searches. Searches of >100 animal spindle assembly factors showed that the genes involved in this process are well conserved in plants, with the exception of two major missing elements: centrosomal components and subunits/regulators of the cytoplasmic dynein complex. We then describe the spindle and phragmoplast assembly mechanisms based on the data obtained from robust gene loss-of-function analyses using RNA interference (RNAi) or mutant plants. Finally, we discuss future research prospects of plant spindles. [ABSTRACT FROM AUTHOR]

The adenomatous polyposis coli (APC) tumor suppressor is multi-functional. APC is known to localize at the centrosome, and in mitotic cells contributes to formation of the mitotic spindle. To test whether APC contributes to nascent microtubule (MT) growth at interphase centrosomes, we employed MT regrowth assays in U2OS cells to measure MT assembly before and after nocodazole treatment and release. We showed that siRNA knockdown of full-length APC delayed both initial MT aster formation and MT elongation/regrowth. In contrast, APC-mutant SW480 cancer cells displayed a defect in MT regrowth that was unaffected by APC knockdown, but which was rescued by reconstitution of full-length APC. Our findings identify APC as a positive regulator of centrosome MT initial assembly and suggest that this process is disrupted by cancer mutations. We confirmed that full-length APC associates with the MT-nucleation factor γ-tubulin, and found that the APC cancer-truncated form (1–1309) also bound to γ-tubulin through APC amino acids 1–453. While binding to γ-tubulin may help target APC to the site of MT nucleation complexes, additional C-terminal sequences of APC are required to stimulate and stabilize MT growth. [ABSTRACT FROM AUTHOR]

Nuclear polarity is characterized by intracytoplasmic nuclear positioning and alignment in the tissue. The mechanisms responsible for maintaining nuclear polarity in normal cells and its disturbance in neoplastic cells are not understood. We studied microtubule-organizing center (MTOC) positioning-mediated nuclear polarity in various normal and neoplastic human tissues, as well as in cultured cells. To visualize the MTOC in cells, gamma-tubulin and pericentrin were immunohistochemically stained by fluorescence and non-fluorescence methods. Position of MTOC in normal and neoplastic tissue was assessed by spatial relationship with nucleus and apico-basal axis. We found MTOC positioning to be related to morphogenesis in various normal and neoplastic human tissues, as well as in cultured cells. MTOC positions were different between two-dimensional cultured isolated cells and three-dimensional cultured gland-formed cells. The MTOC position was specific depending on the cell type in the tissue structure. In particular, glandular and urothelial epithelium had a strong relationship with preservation of nuclear polarity and MTOC positioning. Carcinoma cells showed an irregular position or absence of the MTOC depending on poorer differentiation and higher grade of carcinomas. In conclusion, the position of the MTOC affects regulation of nuclear polarity and morphogenesis of normal and pathological tissue structure. [ABSTRACT FROM AUTHOR]

Objectives: The purpose was to evaluate the association of Aurora A and gamma-tubulin expression with disease characteristics and survival in patients with astrocytoma. Methods: This is a retrospective study of patients who had surgical specimens that were pathologically diagnosed as astrocytoma. The expression level of Aurora A and gamma-tubulin in tumor tissue was evaluated by immunohistochemistry. Clinical information, Karnofsky performance status scale, and survival status of patients were collected. Results: We found that high protein levels of gamma-tubulin or Aurora A were associated with patients 45 years of age, high tumor grade, more advanced non-fully resectable tumors, and poorer survival status. The survival time for patients whose tumors had high gamma-tubulin and Aurora A expression was about 12 months compared with approximately 41 months for patients with low levels of expression of these proteins. Poor patient performance status following resection was also associated with high levels of gamma-tubulin and Aurora A expression. Discussion: The expression levels of gamma-tubulin or Aurora A kinase were associated with patients' age, astrocytoma grade, respectability, as well as patient survival and performance. These findings support the idea that these factors may potentially be important prognostic indicators for patients with astrocytomas. [ABSTRACT FROM AUTHOR]

Y. Chiba, S. Takei, N. Kawamura, Y. Kawaguchi, K. Sasaki, S. Hasegawa-Ishii, A. Furukawa, M. Hosokawa and A. Shimada (2012) Neuropathology and Applied Neurobiology 38, 559-571 Immunohistochemical localization of aggresomal proteins in glial cytoplasmic inclusions in multiple system atrophy Aims: Multiple system atrophy (MSA) is pathologically characterized by the formation of α-synuclein-containing glial cytoplasmic inclusions (GCIs) in oligodendrocytes. However, the mechanisms of GCI formation are not fully understood. Cellular machinery for the formation of aggresomes has been linked to the biogenesis of the Lewy body, a characteristic α-synuclein-containing inclusion of Parkinson's disease and dementia with Lewy bodies. Here, we examined whether GCIs contain the components of aggresomes by immunohistochemistry. Methods: Sections from five patients with MSA were stained immunohistochemically with antibodies against aggresome-related proteins and analysed in comparison with sections from five patients with no neurological disease. We evaluated the presence or absence of aggresome-related proteins in GCIs by double immunofluorescence and immunoelectron microscopy. Results: GCIs were clearly immunolabelled with antibodies against aggresome-related proteins, such as γ-tubulin, histone deacetylase 6 (HDAC6) and 20S proteasome subunits. Neuronal cytoplasmic inclusions (NCIs) were also immunopositive for these aggresome-related proteins. Double immunofluorescence staining and quantitative analysis demonstrated that the majority of GCIs contained these proteins, as well as other aggresome-related proteins, such as Hsp70, Hsp90 and 62-kDa protein/sequestosome 1 (p62/SQSTM1). Immunoelectron microscopy demonstrated immunoreactivities for γ-tubulin and HDAC6 along the fibrils comprising GCIs. Conclusions: Our results indicate that GCIs, and probably NCIs, share at least some characteristics with aggresomes in terms of their protein components. Therefore, GCIs and NCIs may be another manifestation of aggresome-related inclusion bodies observed in neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

The microtubule (MT) cytoskeleton is reorganized during myogenesis as individual myoblasts fuse into multinucleated myotubes. Although this reorganization has long been observed in cell culture, these findings have not been validated during development, and proteins that regulate this process are largely unknown. We have identified a novel postmitotic function for the cytokinesis proteins RacGAP50C (Tumbleweed) and Pavarotti as essential regulators of MT organization during Drosophila myogenesis. We show that the localization of the MT nucleator γ-tubulin changes from diffuse cytoplasmic staining in mononucleated myoblasts to discrete cytoplasmic puncta at the nuclear periphery in multinucleated myoblasts, and that this change in localization depends on RacGAP50C. RacGAP50C and γ-tubulin colocalize at perinuclear sites in myotubes, and in RacGAP50C mutants γ-tubulin remains dispersed throughout the cytoplasm. Furthermore, we show that the mislocalization of RacGAP50C in pavarotti mutants is sufficient to redistribute γ-tubulin to the muscle fiber ends. Finally, myotubes in RacGAP50C mutants have MTs with non-uniform polarity, resulting in multiple guidance errors. Taken together, these findings provide strong evidence that the reorganization of the MT network that has been observed in vitro plays an important role in myotube extension and muscle patterning in vivo, and also identify two molecules crucial for this process. [ABSTRACT FROM AUTHOR]

The relationship between microtubular dynamics, dismantling of pericentriolar components and induction of apoptosis was analysed after exposure of H460 non-small lung cancer cells to anti-mitotic drugs. The microtubule destabilising agent, combretastatin-A4 (CA-4) led to microtubular array disorganization, arrest in mitosis and abnormal metaphases, accompanied by the presence of numerous centrosome-independent “star-like” structures containing tubulin and aggregates of pericentrosomal matrix components like γ-tubulin, pericentrin and ninein, whereas the structural integrity of centrioles was not affected by treatment. On the contrary, in condition of prolonged exposure or high concentrations of CA-4 such aggregates never formed. Treatment with 7.5 nM CA-4, which produced a high frequency “star-like” aggregates, was accompanied by mitotic catastrophe commitment characterized by translocation of the proapoptotic Bim protein to mitochondria activation of caspases-3/9 and DNA fragmentation as a result of either prolonged metaphase arrest or attempt of cells to divide. Drug concentrations which fail to block cells at mitosis were also unable to activate apotosis. A detailed time-course analysis of cell cycle arrest and apoptosis indicated that after CA-4 washout the number of metaphases with “star-like” structures decreased as a function of time and arrested cells proceeded in anaphase. After 4 h, the multiple α- and γ-tubulin aggregates coalesced into two well-defined spindles in a bipolar mitotic spindle organization. Overall, our findings suggest that the maintenance of microtubular integrity plays a relevant role in stabilising the pericentriolar matrix, whose dismantling can be associated with apoptosis after exposure to microtubule depolymerising agents. [ABSTRACT FROM AUTHOR]

Vanadium is an important environmental and industrial pollutant whose concentrations have increased in the last decades. Due to its status as reproductive toxicant and a microtubule damaging agent, the present study investigated by immunohistochemistry the effect of the inhalation of vanadium pentoxide on gamma-tubulin within somatic and testicular germ cells. Male mice inhaled vanadium pentoxide (V2O5) (0.02 M) 1 h/twice a week for 12 weeks. Our results demonstrated that vanadium accumulates in the testes starting with the initial inhalation (24 h), and this pattern remained until the last week of treatment. In general, vanadium was capable of significantly decreasing the percentage of gamma-tubulin in all analyzed testicular cells (Sertoli, Leydig and germ cells) starting with the first week of treatment. For all cell types studied, regression analysis revealed a negative and significant relationship between the percentage of immunopositive cells to gamma-tubulin and exposure time, showing a time dependent response in all cases. Our findings suggest that alterations on this protein might imply changes in microtubule-involved function such as cell division, which in the testes might lead to damage in the spermatogenesis, leading probably to infertility. [ABSTRACT FROM AUTHOR]

Nonreceptor protein tyrosine kinases of the Src family have been shown to play an important role in signal transduction as well as in regulation of microtubule protein interactions. Here we show that γ-tubulin (γ-Tb) in P19 embryonal carcinoma cells undergoing neuronal differentiation is phosphorylated and forms complexes with protein tyrosine kinases of the Src family, Src and Fyn. Elevated expression of both kinases during differentiation corresponded with increased level of proteins phosphorylated on tyrosine. Immunoprecipitation experiments with antibodies against Src, Fyn, γ-tubulin, and with anti-phosphotyrosine antibody revealed that γ-tubulin appeared in complexes with these kinases. In vitro kinase assays showed tyrosine phosphorylation of proteins in γ-tubulin complexes isolated from differentiated cells. Pretreatment of cells with Src family selective tyrosine kinase inhibitor PP2 reduced the amount of phosphorylated γ-tubulin in the complexes. Binding experiments with recombinant SH2 and SH3 domains of Src and Fyn kinases revealed that protein complexes containing γ-tubulin bound to SH2 domains and that these interactions were of SH2-phosphotyrosine type. The combined data suggest that Src family kinases might have an important role in the regulation of γ-tubulin interaction with tubulin dimers or other proteins during neurogenesis. [Copyright &y& Elsevier]

A panel of six anti-peptide antibodies recognizing epitopes in different regions of the γ-tubulin molecule was used for the characterization and localization of γ-tubulin during cell cycle in Leishmania promastigotes. Immunofluorescence microscopy revealed the presence of γ-tubulin in the basal bodies, posterior pole of the cell, and in the flagellum. Furthermore, the antibodies showed punctuate staining in the subpellicular microtubule. This complex localization pattern was observed in both interphase and dividing cells, where staining of posterior poles and the subpellicular corset was more prominent. In posterior poles, γ-tubulin co-distributed with the 210-kDa microtubule-interacting protein and the 57-kDa protein immunodetected with anti-vimentin antibody. Immunogold electron microscopy on thin sections of isolated flagella showed that γ-tubulin was associated with the paraflagellar rod (PFR) that runs adjacent to the axonemal microtubules. Under different extraction conditions, γ-tubulin in Leishmania was found only in insoluble cytoskeletal fractions, in contrast to tubulin dimers that were both in soluble and cytoskeletal pool. Two-dimensional electrophoresis revealed multiple charge variants of γ-tubulin. Posttranslational modifications of Leishmania γ-tubulin might therefore have an important role in the regulation of microtubule nucleation and interaction with other proteins. The complex pattern of γ-tubulin localization and its properties indicate that γ-tubulin in Leishmania might have other function(s) besides microtubule nucleation. [Copyright &y& Elsevier]

Higher plants represent a large group of eukaryotes where centrosomes are absent. The functions of γ-tubulin small complexes (γ-TuSCs) and γ-tubulin ring complexes (γ-TuRCs) in metazoans and fungi in microtubule nucleation are well established and the majority of components found in the complexes are present in plants. However, plant microtubules are also nucleated in a γ-tubulin-dependent but γ-TuRC-independent manner. There is growing evidence that γ-tubulin is a microtubule nucleator without being complexed in γ-TuRC. Fibrillar arrays of γ-tubulin were demonstrated in plant and animal cells and the ability of γ-tubulin to assemble into linear oligomers/polymers was confirmed in vitro for both native and recombinant γ-tubulin. The functions of γ-tubulin as a template for microtubule nucleation or in promoting spontaneous nucleation is outlined. Higher plants represent an excellent model for studies on the role of γ-tubulin in nucleation due to their acentrosomal nature and high abundancy and conservation of γ-tubulin including its intrinsic ability to assemble filaments. The defining scaffolding or sequestration functions of plant γ-tubulin in microtubule organization or in nuclear processes will help our understanding of its cellular roles in eukaryotes. [ABSTRACT FROM AUTHOR]

The present fine structure and anti-tubulin immunofluorescence study deals with evaginations from the cell surface in metaphase I spermatocytes of the firebug Pyrrhocoris apterus (Pyrrhocoridae, Hemiptera). The surface of spermatogonia and prophase spermatocytes was smooth throughout. Only in metaphase I and anaphase I, cytoplasmic threads projected from polar portions of the spermatocytes. In contrast, equatorial portions of these cells possessed a smooth surface. By mid to late telophase, the evaginations were no longer detectable in spermatocytes. Three ideas are at hand to explain the development of polar cytoplasmic evaginations. First, they could represent a membrane reserve used up during spindle elongation in telophase of meiosis. In order to test this idea, spindle structure was analyzed in meiosis I using simultaneously antibodies to β-tubulin and γ-tubulin. γ-Tubulin represents a tubulin isoform prevalent in centrosomes. The observations showed that spindle elongation was not very prominent in meiosis of the bug. Although it cannot be ruled out, the formation of a polar membrane reserve prior to spindle elongation is not a likely explanation for the evaginations from the cell surface. Second, the development of surface extensions could bring about increased exchange of metabolites during a particularly active stage of meiosis. Third, the polar evaginations could be an inadvertent product of the aster microtubules protruding towards the plasma membrane. [ABSTRACT FROM AUTHOR]

Production of a protein of interest in bacteria and its purification from bacterial lysates are valuable tools for the purification of larger amounts of recombinant proteins. The low cost of culturing, and the rapid cell growth of bacteria make this host a good choice for protein production, but the folding and function of the purified protein might be altered due to the production of a eukaryotic protein in a prokaryotic host. Here, we provide a purification method for the purification of gamma (γ)-tubulin (TUBG) from soluble fractions of Escherichia (E.) coli lysates using affinity tags.•This protocol describes a method that purifies soluble GST-TUBG1 from bacteria.•Of the three tested induction conditions, the highest yield of recombinant GST-TUBG1 was obtained after the induction of E. coli with isopropyl-D-1-thiogalactopyranoside (IPTG) for 1 h at 37 °C followed by overnight incubation at room temperature.•In comparison with other methodologies (Hoog et al., 2011), the technique described here retrieves larger amounts of recombinant TUBG1 from small-scale expression cultures., (© 2021 The Authors. Published by Elsevier B.V.)