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.

• Klíčová slova
• Arabidopsis thaliana, AtTPX2, Aurora kinase, Ran., fibres, importin, microtubules, nuclei, γ-tubulin,
• MeSH
• apoptóza MeSH
• Arabidopsis cytologie   enzymologie   metabolismus   MeSH
• buněčné jádro metabolismus   MeSH
• centrozom metabolismus   MeSH
• chromatin metabolismus   MeSH
• jaderný obal metabolismus   MeSH
• karyoferiny metabolismus   MeSH
• kinasy aurora metabolismus   MeSH
• mikrotubuly metabolismus   MeSH
• mitóza MeSH
• počítačová simulace MeSH
• proteiny asociované s mikrotubuly metabolismus   MeSH
• proteiny huseníčku metabolismus   MeSH
• rostlinné buňky metabolismus   MeSH
• subcelulární frakce metabolismus   MeSH
• transport proteinů MeSH
• tubulin metabolismus   MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• zobrazování trojrozměrné MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromatin MeSH
• karyoferiny MeSH
• kinasy aurora MeSH
• proteiny asociované s mikrotubuly MeSH
• proteiny huseníčku MeSH
• TPX2 protein, Arabidopsis MeSH Prohlížeč
• tubulin MeSH
• zelené fluorescenční proteiny MeSH

The nodulin/glutamine synthetase-like protein (NodGS) that we identified proteomically in Arabidopsis thaliana is a fusion protein composed of an N-terminal amidohydrolase domain that shares homology with nodulins and a C-terminal domain of prokaryotic glutamine synthetase type I. The protein is homologous to the FluG protein, a morphogenetic factor in fungi. Although genes encoding NodGS homologues are present in many plant genomes, their products have not yet been characterized. The Arabidopsis NodGS was present in an oligomeric form of ~700-kDa, mainly in the cytosol, and to a lesser extent in the microsomal membrane fraction. The oligomeric NodGS was incorporated into large heterogeneous protein complexes >700 kDa and partially co-immunoprecipitated with γ-tubulin. In situ and in vivo microscopic analyses revealed a NodGS signal in the cytoplasm, with endomembranes, particularly in the perinuclear area. NodGS had no detectable glutamine synthetase activity. Downregulation of NodGS by RNAi resulted in plants with a short main root, reduced meristematic activity and disrupted development of the root cap. Y2H analysis and publicly available microarray data indicated a role for NodGS in biotic stress signalling. We found that flagellin enhanced the expression of the NodGS protein, which was then preferentially localized in the nuclear periphery. Our results point to a role for NodGS in root morphogenesis and microbial elicitation. These data might help in understanding the family of NodGS/FluG-like fusion genes that are widespread in prokaryotes, fungi and plants.

• MeSH
• Arabidopsis genetika   růst a vývoj   metabolismus   MeSH
• flagelin genetika   metabolismus   MeSH
• glutaminsynthetasa genetika   metabolismus   fyziologie   MeSH
• kořeny rostlin genetika   růst a vývoj   metabolismus   MeSH
• membránové proteiny genetika   metabolismus   fyziologie   MeSH
• morfogeneze fyziologie   MeSH
• proteiny huseníčku genetika   metabolismus   fyziologie   MeSH
• regulace genové exprese u rostlin MeSH
• regulátory růstu rostlin metabolismus   MeSH
• rostlinné geny MeSH
• rostlinné proteiny genetika   metabolismus   fyziologie   MeSH
• signální transdukce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• flagelin MeSH
• glutaminsynthetasa MeSH
• membránové proteiny MeSH
• nodulin MeSH Prohlížeč
• proteiny huseníčku MeSH
• regulátory růstu rostlin MeSH
• rostlinné proteiny 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 anatomie a histologie   cytologie   metabolismus   MeSH
• buněčné jádro fyziologie   MeSH
• down regulace MeSH
• fenotyp MeSH
• kořeny rostlin cytologie   růst a vývoj   metabolismus   MeSH
• listy rostlin cytologie   růst a vývoj   metabolismus   MeSH
• mikrotubuly metabolismus   ultrastruktura   MeSH
• mitóza fyziologie   MeSH
• molekulární sekvence - údaje MeSH
• proteiny huseníčku genetika   fyziologie   MeSH
• RNA interference MeSH
• tubulin genetika   fyziologie   MeSH
• zvětšování buněk MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny huseníčku 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
• antinukleární protilátky genetika   metabolismus   MeSH
• Arabidopsis metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• cytosol metabolismus   MeSH
• dimerizace MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• fluorescenční protilátková technika MeSH
• mikrotubuly metabolismus   MeSH
• mikrozomy metabolismus   MeSH
• mitóza fyziologie   MeSH
• precipitinové testy MeSH
• proteiny huseníčku metabolismus   MeSH
• tubulin chemie   imunologie   metabolismus   MeSH
• vazba proteinů MeSH
• Vicia faba metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antinukleární protilátky MeSH
• proteiny huseníčku MeSH
• tubulin MeSH

Neither the molecular mechanism by which plant microtubules nucleate in the cytoplasm nor the organization of plant mitotic spindles, which lack centrosomes, is well understood. Here, using immunolocalization and cell fractionation techniques, we provide evidence that gamma-tubulin, a universal component of microtubule organizing centers, is present in both the cytoplasm and the nucleus of plant cells. The amount of gamma-tubulin in nuclei increased during the G(2) phase, when cells are synchronized or sorted for particular phases of the cell cycle. gamma-Tubulin appeared on prekinetochores before preprophase arrest caused by inhibition of the cyclin-dependent kinase and before prekinetochore labeling of the mitosis-specific phosphoepitope MPM2. The association of nuclear gamma-tubulin with chromatin displayed moderately strong affinity, as shown by its release after DNase treatment and by using extraction experiments. Subcellular compartmentalization of gamma-tubulin might be an important factor in the organization of plant-specific microtubule arrays and acentriolar mitotic spindles.

• MeSH
• buněčné jádro chemie   MeSH
• buněčný cyklus MeSH
• centrioly MeSH
• Fabaceae chemie   metabolismus   MeSH
• fluorescenční protilátková technika MeSH
• konfokální mikroskopie MeSH
• léčivé rostliny MeSH
• mitóza * MeSH
• rostliny chemie   metabolismus   MeSH
• tubulin metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• tubulin MeSH