Microtubules composed of αβ-tubulin dimers are dynamic cytoskeletal polymers that play key roles in essential cellular processes such as cell division, organelle positioning, intracellular transport, and cell migration. γ-Tubulin is a highly conserved member of the tubulin family that is required for microtubule nucleation. γ-Tubulin, together with its associated proteins, forms the γ-tubulin ring complex (γ-TuRC), that templates microtubules. Here we review recent advances in the structure of γ-TuRC, its activation, and centrosomal recruitment. This provides new mechanistic insights into the molecular mechanism of microtubule nucleation. Accumulating data suggest that γ-tubulin also has other, less well understood functions. We discuss emerging evidence that γ-tubulin can form oligomers and filaments, has specific nuclear functions, and might be involved in centrosomal cross-talk between microtubules and microfilaments.

• Keywords
• microtubule nucleation, αβ-tubulin dimer, γ-tubulin functions, γ-tubulin isotypes, γ-tubulin ring complexes (γ-TuRC),
• Publication type
• Journal Article MeSH
• Review MeSH

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.

• Keywords
• fibrillar arrays, gamma-tubulin, gamma-tubulin complexes, microtubules, nucleation, plants, sequestration, signaling,
• MeSH
• Cells metabolism   MeSH
• Centrosome metabolism   MeSH
• Humans MeSH
• Plants metabolism   MeSH
• Amino Acid Sequence MeSH
• Tubulin chemistry   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Tubulin MeSH

Microtubules of all eukaryotic cells are formed by α- and β-tubulin heterodimers. In addition to the well known cytoplasmic tubulins, a subpopulation of tubulin can occur in the nucleus. So far, the potential function of nuclear tubulin has remained elusive. In this work, we show that α- and β-tubulins of various organisms contain multiple conserved nuclear export sequences, which are potential targets of the Exportin 1/CRM1 pathway. We demonstrate exemplarily that these NES motifs are sufficient to mediate export of GFP as model cargo and that this export can be inhibited by leptomycin B, an inhibitor of the Exportin 1/CRM1 pathway. Likewise, leptomycin B causes accumulation of GFP-tagged tubulin in interphase nuclei, in both plant and animal model cells. Our analysis of nuclear tubulin content supports the hypothesis that an important function of nuclear tubulin export is the exclusion of tubulin from interphase nuclei, after being trapped by nuclear envelope reassembly during telophase.

• MeSH
• Active Transport, Cell Nucleus physiology   MeSH
• Cell Nucleus metabolism   MeSH
• Cell Line MeSH
• Cytoplasm metabolism   MeSH
• Eukaryotic Cells metabolism   MeSH
• Karyopherins metabolism   MeSH
• Humans MeSH
• Microtubules metabolism   MeSH
• Exportin 1 Protein MeSH
• Receptors, Cytoplasmic and Nuclear metabolism   MeSH
• Nicotiana metabolism   MeSH
• Protein Transport physiology   MeSH
• Tubulin metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Karyopherins MeSH
• Receptors, Cytoplasmic and Nuclear MeSH
• Tubulin MeSH

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.

• Keywords
• SUN proteins, filaments, gamma-tubulin, lamins, mechanosensing, nuclear functions, nucleation,
• MeSH
• Cell Nucleus metabolism   MeSH
• Cell Cycle MeSH
• Nuclear Proteins metabolism   MeSH
• Nuclear Envelope metabolism   MeSH
• Humans MeSH
• Microtubules metabolism   MeSH
• Tubulin metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Nuclear Proteins MeSH
• Tubulin MeSH

The microtubule cytoskeleton is critically important for spatio-temporal organization of eukaryotic cells. The nucleation of new microtubules is typically restricted to microtubule organizing centers (MTOCs) and requires γ-tubulin that assembles into multisubunit complexes of various sizes. γ-Tubulin ring complexes (TuRCs) are efficient microtubule nucleators and are associated with large number of targeting, activating and modulating proteins. γ-Tubulin-dependent nucleation of microtubules occurs both from canonical MTOCs, such as spindle pole bodies and centrosomes, and additional sites such as Golgi apparatus, nuclear envelope, plasma membrane-associated sites, chromatin and surface of pre-existing microtubules. Despite many advances in structure of γ-tubulin complexes and characterization of γTuRC interacting factors, regulatory mechanisms of microtubule nucleation are not fully understood. Here, we review recent work on the factors and regulatory mechanisms that are involved in centrosomal and non-centrosomal microtubule nucleation.

• Keywords
• Centrosomes, Microtubule nucleation, Microtubule-organizing centers, Non-centrosomal nucleation sites, Spindle pole bodies, γ-Tubulin complexes,
• MeSH
• Centrosome metabolism   MeSH
• Golgi Apparatus metabolism   MeSH
• Nuclear Envelope metabolism   MeSH
• Humans MeSH
• Microtubules metabolism   MeSH
• Multiprotein Complexes metabolism   MeSH
• Spindle Pole Bodies metabolism   MeSH
• Microtubule-Associated Proteins metabolism   MeSH
• Tubulin metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Multiprotein Complexes MeSH
• Microtubule-Associated Proteins MeSH
• Tubulin MeSH

TPX2 performs multiple roles in microtubule organization. Previously, it was shown that plant AtTPX2 binds AtAurora1 kinase and colocalizes with microtubules in a cell cycle-specific manner. To elucidate the function of TPX2 further, this work analysed Arabidopsis cells overexpressing AtTPX2-GFP. Distinct arrays of bundled microtubules, decorated with AtTPX2-GFP, were formed in the vicinity of the nuclear envelope and in the nuclei of overexpressing cells. The microtubular arrays showed reduced sensitivity to anti-microtubular drugs. TPX2-mediated formation of nuclear/perinuclear microtubular arrays was not specific for the transition to mitosis and occurred independently of Aurora kinase. The fibres were not observed in cells with detectable programmed cell death and, in this respect, they differed from TPX2-dependent microtubular assemblies functioning in mammalian apoptosis. Colocalization and co-purification data confirmed the interaction of importin with AtTPX2-GFP. In cells with nuclear foci of overexpressed AtTPX2-GFP, strong nuclear signals for Ran and importin diminished when microtubular arrays were assembled. This observation suggests that TPX2-mediated microtubule formation might be triggered by a Ran cycle. Collectively, the data suggest that in the acentrosomal plant cell, in conjunction with importin, overexpressed AtTPX2 reinforces microtubule formation in the vicinity of chromatin and the nuclear envelope.

• Keywords
• Arabidopsis thaliana, AtTPX2, Aurora kinase, Ran., fibres, importin, microtubules, nuclei, γ-tubulin,
• MeSH
• Apoptosis MeSH
• Arabidopsis cytology   enzymology   metabolism   MeSH
• Cell Nucleus metabolism   MeSH
• Centrosome metabolism   MeSH
• Chromatin metabolism   MeSH
• Nuclear Envelope metabolism   MeSH
• Karyopherins metabolism   MeSH
• Aurora Kinases metabolism   MeSH
• Microtubules metabolism   MeSH
• Mitosis MeSH
• Computer Simulation MeSH
• Microtubule-Associated Proteins metabolism   MeSH
• Arabidopsis Proteins metabolism   MeSH
• Plant Cells metabolism   MeSH
• Subcellular Fractions metabolism   MeSH
• Protein Transport MeSH
• Tubulin metabolism   MeSH
• Green Fluorescent Proteins metabolism   MeSH
• Imaging, Three-Dimensional MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin MeSH
• Karyopherins MeSH
• Aurora Kinases MeSH
• Microtubule-Associated Proteins MeSH
• Arabidopsis Proteins MeSH
• TPX2 protein, Arabidopsis MeSH Browser
• Tubulin MeSH
• Green Fluorescent Proteins MeSH

Concurrently with cold-induced disintegration of microtubular structures in the cytoplasm, gradual tubulin accumulation was observed in a progressively growing proportion of interphase nuclei in tobacco BY-2 cells. This intranuclear tubulin disappeared upon rewarming. Simultaneously, new microtubules rapidly emerged from the nuclear periphery and reconstituted new cortical arrays, as was shown by immunofluorescence. A rapid exclusion of tubulin from the nucleus during rewarming was also observed in vivo in cells expressing GFP-tubulin. Nuclei were purified from cells that expressed GFP fused to an endoplasmic-reticulum retention signal (BY-2-mGFP5-ER), and green-fluorescent protein was used as a diagnostic marker to confirm that the nuclear fraction was not contaminated by nuclear-envelope proteins. These purified, GFP-free nuclei contained tubulin when isolated from cold-treated cells, whereas control nuclei were void of tubulin. Furthermore, highly conserved putative nuclear-export sequences were identified in tubulin sequences. These results led us to interpret the accumulation of tubulin in interphasic nuclei, as well as its rapid nuclear export, in the context of ancient intranuclear tubulin function during the cell cycle progression.

• MeSH
• Cell Nucleus metabolism   MeSH
• Microtubules metabolism   MeSH
• Molecular Sequence Data MeSH
• Cold Temperature * MeSH
• Amino Acid Sequence MeSH
• Nuclear Export Signals MeSH
• Nicotiana cytology   metabolism   MeSH
• Tubulin chemistry   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Nuclear Export Signals MeSH
• Tubulin MeSH

Gamma-tubulin is required for microtubule (MT) nucleation at MT organizing centers such as centrosomes or spindle pole bodies, but little is known about its noncentrosomal functions. We conditionally downregulated gamma-tubulin by inducible expression of RNA interference (RNAi) constructs in Arabidopsis thaliana. Almost complete RNAi depletion of gamma-tubulin led to the absence of MTs and was lethal at the cotyledon stage. After induction of RNAi expression, gamma-tubulin was gradually depleted from both cytoplasmic and microsomal fractions. In RNAi plants with partial loss of gamma-tubulin, MT recovery after drug-induced depolymerization was impaired. Similarly, immunodepletion of gamma-tubulin from Arabidopsis extracts severely compromised in vitro polymerization of MTs. Reduction of gamma-tubulin protein levels led to randomization and bundling of cortical MTs. This finding indicates that MT-bound gamma-tubulin is part of a cortical template guiding the microtubular network and is essential for MT nucleation. Furthermore, we found that cells with decreased levels of gamma-tubulin could progress through mitosis, but cytokinesis was strongly affected. Stepwise diminution of gamma-tubulin allowed us to reveal roles for MT nucleation in plant development, such as organization of cell files, anisotropic and polar tip growth, and stomatal patterning. Some of these functions of gamma-tubulin might be independent of MT nucleation.

• MeSH
• Arabidopsis anatomy & histology   cytology   metabolism   MeSH
• Cell Nucleus physiology   MeSH
• Down-Regulation MeSH
• Phenotype MeSH
• Plant Roots cytology   growth & development   metabolism   MeSH
• Plant Leaves cytology   growth & development   metabolism   MeSH
• Microtubules metabolism   ultrastructure   MeSH
• Mitosis physiology   MeSH
• Molecular Sequence Data MeSH
• Arabidopsis Proteins genetics   physiology   MeSH
• RNA Interference MeSH
• Tubulin genetics   physiology   MeSH
• Cell Enlargement MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Arabidopsis Proteins MeSH
• Tubulin MeSH

gamma-Tubulin is assumed to participate in microtubule nucleation in acentrosomal plant cells, but the underlying molecular mechanisms are still unknown. Here, we show that gamma-tubulin is present in protein complexes of various sizes and different subcellular locations in Arabidopsis and fava bean. Immunoprecipitation experiments revealed an association of gamma-tubulin with alphabeta-tubulin dimers. gamma-Tubulin cosedimented with microtubules polymerized in vitro and localized along their whole length. Large gamma-tubulin complexes resistant to salt treatment were found to be associated with a high-speed microsomal fraction. Blue native electrophoresis of detergent-solubilized microsomes showed that the molecular mass of the complexes was >1 MD. Large gamma-tubulin complexes were active in microtubule nucleation, but nucleation activity was not observed for the smaller complexes. Punctate gamma-tubulin staining was associated with microtubule arrays, accumulated with short kinetochore microtubules interacting in polar regions with membranes, and localized in the vicinity of nuclei and in the area of cell plate formation. Our results indicate that the association of gamma-tubulin complexes with dynamic membranes might ensure the flexibility of noncentrosomal microtubule nucleation. Moreover, the presence of other molecular forms of gamma-tubulin suggests additional roles for this protein species in microtubule organization.

• MeSH
• Antibodies, Antinuclear genetics   metabolism   MeSH
• Arabidopsis metabolism   MeSH
• Cell Membrane metabolism   MeSH
• Cytosol metabolism   MeSH
• Dimerization MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Fluorescent Antibody Technique MeSH
• Microtubules metabolism   MeSH
• Microsomes metabolism   MeSH
• Mitosis physiology   MeSH
• Precipitin Tests MeSH
• Arabidopsis Proteins metabolism   MeSH
• Tubulin chemistry   immunology   metabolism   MeSH
• Protein Binding MeSH
• Vicia faba metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antibodies, Antinuclear MeSH
• Arabidopsis Proteins MeSH
• Tubulin MeSH