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.

• Klíčová slova
• microtubule nucleation, αβ-tubulin dimer, γ-tubulin functions, γ-tubulin isotypes, γ-tubulin ring complexes (γ-TuRC),
• Publikační typ
• časopisecké články MeSH
• přehledy 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.

• Klíčová slova
• fibrillar arrays, gamma-tubulin, gamma-tubulin complexes, microtubules, nucleation, plants, sequestration, signaling,
• MeSH
• buňky metabolismus   MeSH
• centrozom metabolismus   MeSH
• lidé MeSH
• rostliny metabolismus   MeSH
• sekvence aminokyselin MeSH
• tubulin chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• 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.

• Klíčová slova
• SUN proteins, filaments, gamma-tubulin, lamins, mechanosensing, nuclear functions, nucleation,
• MeSH
• buněčné jádro metabolismus   MeSH
• buněčný cyklus MeSH
• jaderné proteiny metabolismus   MeSH
• jaderný obal metabolismus   MeSH
• lidé MeSH
• mikrotubuly metabolismus   MeSH
• tubulin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• jaderné proteiny MeSH
• tubulin MeSH

Plants employ acentrosomal mechanisms to organize cortical microtubule arrays essential for cell growth and differentiation. Using structured illumination microscopy (SIM) adopted for the optimal documentation of Arabidopsis (Arabidopsis thaliana) hypocotyl epidermal cells, dynamic cortical microtubules labeled with green fluorescent protein fused to the microtubule-binding domain of the mammalian microtubule-associated protein MAP4 and with green fluorescent protein-fused to the alpha tubulin6 were comparatively recorded in wild-type Arabidopsis plants and in the mitogen-activated protein kinase mutant mpk4 possessing the former microtubule marker. The mpk4 mutant exhibits extensive microtubule bundling, due to increased abundance and reduced phosphorylation of the microtubule-associated protein MAP65-1, thus providing a very useful genetic tool to record intrabundle microtubule dynamics at the subdiffraction level. SIM imaging revealed nano-sized defects in microtubule bundling, spatially resolved microtubule branching and release, and finally allowed the quantification of individual microtubules within cortical bundles. Time-lapse SIM imaging allowed the visualization of subdiffraction, short-lived excursions of the microtubule plus end, and dynamic instability behavior of both ends during free, intrabundle, or microtubule-templated microtubule growth and shrinkage. Finally, short, rigid, and nondynamic microtubule bundles in the mpk4 mutant were observed to glide along the parent microtubule in a tip-wise manner. In conclusion, this study demonstrates the potential of SIM for superresolution time-lapse imaging of plant cells, showing unprecedented details accompanying microtubule dynamic organization.

• MeSH
• Arabidopsis metabolismus   MeSH
• epidermis rostlin cytologie   metabolismus   MeSH
• hypokotyl cytologie   metabolismus   MeSH
• konfokální mikroskopie MeSH
• mikroskopie metody   MeSH
• mikrotubuly metabolismus   MeSH
• mutace genetika   MeSH
• osvětlení * MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny huseníčku MeSH
• rekombinantní fúzní proteiny MeSH
• zelené fluorescenční proteiny 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.

• 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

BACKGROUND: RanBPM (Ran-binding protein in the microtubule-organizing centre) was originally reported as a centrosome-associated protein in human cells. However, RanBPM protein containing highly conserved SPRY, LisH, CTLH and CRA domains is currently considered as a scaffolding protein with multiple cellular functions. A plant homologue of RanBPM has not yet been characterized. RESULTS: Based on sequence similarity, we identified a homologue of the human RanBPM in Arabidopsis thaliana. AtRanBPM protein has highly conserved SPRY, LisH, CTLH and CRA domains. Cell fractionation showed that endogenous AtRanBPM or expressed GFP-AtRanBPM are mainly cytoplasmic proteins with only a minor portion detectable in microsomal fractions. AtRanBPM was identified predominantly in the form of soluble cytoplasmic complexes ~230-500 kDa in size. Immunopurification of AtRanBPM followed by mass spectrometric analysis identified proteins containing LisH and CRA domains; LisH, CRA, RING-U-box domains and a transducin/WD40 repeats in a complex with AtRanBPM. Homologues of identified proteins are known to be components of the C-terminal to the LisH motif (CTLH) complexes in humans and budding yeast. Microscopic analysis of GFP-AtRanBPM in vivo and immunofluorescence localization of endogenous AtRanBPM protein in cultured cells and seedlings of Arabidopsis showed mainly cytoplasmic and nuclear localization. Absence of colocalization with γ-tubulin was consistent with the biochemical data and suggests another than a centrosomal role of the AtRanBPM protein. CONCLUSION: We showed that as yet uncharacterized Arabidopsis RanBPM protein physically interacts with LisH-CTLH domain-containing proteins. The newly identified high molecular weight cytoplasmic protein complexes of AtRanBPM showed homology with CTLH types of complexes described in mammals and budding yeast. Although the exact functions of the CTLH complexes in scaffolding of protein degradation, in protein interactions and in signalling from the periphery to the cell centre are not yet fully understood, structural conservation of the complexes across eukaryotes suggests their important biological role.

• MeSH
• adaptorové proteiny signální transdukční genetika   metabolismus   MeSH
• Arabidopsis chemie   genetika   metabolismus   MeSH
• cytoskeletální proteiny genetika   metabolismus   MeSH
• Eukaryota chemie   klasifikace   genetika   MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• konzervovaná sekvence MeSH
• lidé MeSH
• molekulární evoluce * MeSH
• molekulární sekvence - údaje MeSH
• proteiny huseníčku chemie   genetika   metabolismus   MeSH
• rostliny chemie   klasifikace   genetika   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• cytoskeletální proteiny MeSH
• jaderné proteiny MeSH
• proteiny huseníčku MeSH
• Ran binding protein 9 MeSH Prohlížeč

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