γ-tubulin functions Dotaz Zobrazit nápovědu
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.
- 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
γ-Tubulins are highly conserved members of the tubulin superfamily essential for microtubule nucleation. Humans possess 2 γ-tubulin genes. It is thought that γ-tubulin-1 represents a ubiquitous isotype, whereas γ-tubulin-2 is found predominantly in the brain, where it may be endowed with divergent functions beyond microtubule nucleation. The molecular basis of the purported functional differences between γ-tubulins is unknown. We report discrimination of human γ-tubulins according to their electrophoretic and immunochemical properties. In vitro mutagenesis revealed that the differences in electrophoretic mobility originate in the C-terminal regions of the γ-tubulins. Using epitope mapping, we discovered mouse monoclonal antibodies that can discriminate between human γ-tubulin isotypes. Real time quantitative RT-PCR and 2-dimensional-PAGE showed that γ-tubulin-1 is the dominant isotype in fetal neurons. Although γ-tubulin-2 accumulates in the adult brain, γ-tubulin-1 remains the major isotype in various brain regions. Localization of γ-tubulin-1 in mature neurons was confirmed by immunohistochemistry and immunofluorescence microscopy on clinical samples and tissue microarrays. Differentiation of SH-SY5Y human neuroblastoma cells by all-trans retinoic acid, or oxidative stress induced by mitochondrial inhibitors, resulted in upregulation of γ-tubulin-2, whereas the expression of γ-tubulin-1 was unchanged. Fractionation experiments and immunoelectron microscopy revealed an association of γ-tubulins with mitochondrial membranes. These data indicate that in the face of predominant γ-tubulin-1 expression, the accumulation of γ-tubulin-2 in mature neurons and neuroblastoma cells during oxidative stress may denote a prosurvival role of γ-tubulin-2 in neurons.-Dráberová, E., Sulimenko, V., Vinopal, S., Sulimenko, T., Sládková, V., D'Agostino, L., Sobol, M., Hozák, P., Křen, L., Katsetos, C. D., Dráber, P. Differential expression of human γ-tubulin isotypes during neuronal development and oxidative stress points to γ-tubulin-2 prosurvival function.
- MeSH
- lidé MeSH
- mikrotubuly metabolismus MeSH
- neuroblastom metabolismus MeSH
- neurogeneze fyziologie MeSH
- neurony metabolismus MeSH
- oxidační stres fyziologie MeSH
- tubulin metabolismus MeSH
- vývojová regulace genové exprese fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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.
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
γ-Tubulin is the key protein for microtubule nucleation. Duplication of the γ-tubulin gene occurred several times during evolution, and in mammals γ-tubulin genes encode proteins which share ∼97% sequence identity. Previous analysis of Tubg1 and Tubg2 knock-out mice has suggested that γ-tubulins are not functionally equivalent. Tubg1 knock-out mice died at the blastocyst stage, whereas Tubg2 knock-out mice developed normally and were fertile. It was proposed that γ-tubulin 1 represents ubiquitous γ-tubulin, while γ-tubulin 2 may have some specific functions and cannot substitute for γ-tubulin 1 deficiency in blastocysts. The molecular basis of the suggested functional difference between γ-tubulins remains unknown. Here we show that exogenous γ-tubulin 2 is targeted to centrosomes and interacts with γ-tubulin complex proteins 2 and 4. Depletion of γ-tubulin 1 by RNAi in U2OS cells causes impaired microtubule nucleation and metaphase arrest. Wild-type phenotype in γ-tubulin 1-depleted cells is restored by expression of exogenous mouse or human γ-tubulin 2. Further, we show at both mRNA and protein levels using RT-qPCR and 2D-PAGE, respectively, that in contrast to Tubg1, the Tubg2 expression is dramatically reduced in mouse blastocysts. This indicates that γ-tubulin 2 cannot rescue γ-tubulin 1 deficiency in knock-out blastocysts, owing to its very low amount. The combined data suggest that γ-tubulin 2 is able to nucleate microtubules and substitute for γ-tubulin 1. We propose that mammalian γ-tubulins are functionally redundant with respect to the nucleation activity.
- MeSH
- časové faktory MeSH
- down regulace MeSH
- embryonální vývoj genetika MeSH
- implantace embrya MeSH
- intracelulární prostor metabolismus MeSH
- lidé MeSH
- mikrotubuly metabolismus MeSH
- mitóza genetika MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- protein - isoformy nedostatek genetika metabolismus MeSH
- transport proteinů MeSH
- tubulin nedostatek genetika metabolismus MeSH
- vývojová regulace genové exprese MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
γ-Tubulin is essential for microtubule nucleation and also plays less understood roles in nuclear and cell-cycle-related functions. High abundancy of γ-tubulin in acentrosomal Arabidopsis cells facilitated purification and biochemical characterization of large molecular species of γ-tubulin. TEM, fluorescence, and atomic force microscopy of purified high molecular γ-tubulin forms revealed the presence of linear filaments with a double protofilament substructure, filament bundles and aggregates. Filament formation from highly purified γ-tubulin free of γ-tubulin complex proteins (GCPs) was demonstrated for both plant and human γ-tubulin. Moreover, γ-tubulin associated with porcine brain microtubules formed oligomers. Experimental evidence on the intrinsic ability of γ-tubulin to oligomerize/polymerize was supported by conservation of α- and β-tubulin interfaces for longitudinal and lateral interactions for γ-tubulins. STED (stimulated emission depletion) microscopy of Arabidopsis cells revealed fine, short γ-tubulin fibrillar structures enriched on mitotic microtubular arrays that accumulated at polar regions of acentrosomal spindles and the outer nuclear envelope before mitosis, and were also present in nuclei. Fine fibrillar structures of γ-tubulin representing assemblies of higher order were localized in cell-cycle-dependent manner at sites of dispersed γ-tubulin location in acentrosomal plant cells as well as at sites of local γ-tubulin enrichment after drug treatment. Our findings that γ-tubulin preserves the capability of prokaryotic tubulins to self-organize into filaments assembling by lateral interaction into bundles/clusters help understanding of the relationship between structure and multiple cellular functions of this protein species and suggest that besides microtubule nucleation and organization, γ-tubulin may also have scaffolding or sequestration functions.
- MeSH
- Arabidopsis chemie genetika MeSH
- cytoskelet chemie genetika MeSH
- mikrofilamenta chemie genetika ultrastruktura MeSH
- mikrotubuly chemie genetika MeSH
- mitóza genetika MeSH
- polymerizace MeSH
- proteinové agregáty genetika MeSH
- proteiny asociované s mikrotubuly chemie genetika MeSH
- tubulin chemie genetika ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
γ-Tubulin is assumed to be a typical cytosolic protein necessary for nucleation of microtubules from microtubule organizing centers. Using immunolocalization and cell fractionation techniques in combination with siRNAi and expression of FLAG-tagged constructs, we have obtained evidence that γ-tubulin is also present in nucleoli of mammalian interphase cells of diverse cellular origins. Immunoelectron microscopy has revealed γ-tubulin localization outside fibrillar centers where transcription of ribosomal DNA takes place. γ-Tubulin was associated with nucleolar remnants after nuclear envelope breakdown and could be translocated to nucleoli during mitosis. Pretreatment of cells with leptomycin B did not affect the distribution of nuclear γ-tubulin, making it unlikely that rapid active transport via nuclear pores participates in the transport of γ-tubulin into the nucleus. This finding was confirmed by heterokaryon assay and time-lapse imaging of photoconvertible protein Dendra2 tagged to γ-tubulin. Immunoprecipitation from nuclear extracts combined with mass spectrometry revealed an association of γ-tubulin with tumor suppressor protein C53 located at multiple subcellular compartments including nucleoli. The notion of an interaction between γ-tubulin and C53 was corroborated by pull-down and co-immunoprecipitation experiments. Overexpression of γ-tubulin antagonized the inhibitory effect of C53 on DNA damage G(2) /M checkpoint activation. The combined results indicate that aside from its known role in microtubule nucleation, γ-tubulin may also have nuclear-specific function(s).
- MeSH
- astrocyty metabolismus MeSH
- buněčné jadérko metabolismus MeSH
- buněčné jádro metabolismus MeSH
- časosběrné zobrazování MeSH
- fluorescenční protilátková technika MeSH
- glioblastom metabolismus MeSH
- hmotnostní spektrometrie MeSH
- imunoelektronová mikroskopie MeSH
- imunoprecipitace MeSH
- intracelulární signální peptidy a proteiny metabolismus MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- mikrotubuly metabolismus MeSH
- mitóza fyziologie MeSH
- nádorové buněčné linie MeSH
- nádory mozku metabolismus MeSH
- proteiny nervové tkáně metabolismus MeSH
- transport proteinů fyziologie MeSH
- tubulin metabolismus MeSH
- tumor supresorové geny MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Highly conserved α- and β-tubulin heterodimers assemble into dynamic microtubules and perform multiple important cellular functions such as structural support, pathway for transport and force generation in cell division. Tubulin exists in different forms of isotypes expressed by specific genes with spatially- and temporally-regulated expression levels. Some tubulin isotypes are differentially expressed in normal and neoplastic cells, providing a basis for cancer chemotherapy drug development. Moreover, specific tubulin isotypes are overexpressed and localized in the nuclei of cancer cells and/or show bioenergetic functions through the regulation of the permeability of mitochondrial ion channels. It has also become clear that tubulin isotypes are involved in multiple cellular functions without being incorporated into microtubule structures. Understanding the mutations of tubulin isotypes specifically expressed in tumors and their post-translational modifications might help to identify precise molecular targets for the design of novel anti-microtubular drugs. Knowledge of tubulin mutations present in tubulinopathies brings into focus cellular functions of tubulin in brain pathologies such as Alzheimer's disease. Uncovering signaling pathways which affect tubulin functions during antigen-mediated activation of mast cells presents a major challenge in developing new strategies for the treatment of inflammatory and allergic diseases. γ-tubulin, a conserved member of the eukaryotic tubulin superfamily specialized for microtubule nucleation is a target of cell cycle and stress signaling. Besides its microtubule nucleation role, γ-tubulin functions in nuclear and cell cycle related processes. This special issue "Tubulin: Structure, Functions and Roles in Disease" contains eight articles, five of which are original research papers and three are review papers that cover diverse areas of tubulin biology and functions under normal and pathological conditions.
- MeSH
- Alzheimerova nemoc genetika metabolismus patologie MeSH
- lidé MeSH
- mikrotubuly genetika metabolismus patologie MeSH
- mutace MeSH
- nádorové proteiny genetika metabolismus MeSH
- nádory genetika metabolismus MeSH
- protein - isoformy MeSH
- tubulin genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
- úvodní články MeSH
- úvodníky MeSH
Microtubules represent cytoplasmic structures that are indispensable for the maintenance of cell morphology and motility generation. Due to their regular structural organization, microtubules have become of great interest for preparation of in vitro nanotransport systems. However, tubulin, the major building protein of microtubules, is a thermolabile protein and is usually stored at -80 degrees C to preserve its conformation and polymerization properties. Here we describe a novel method for freeze-drying of assembly-competent tubulin in the presence of a nonreducing sugar trehalose. Even after prolonged storage at ambient temperature, rehydrated tubulin is capable of binding antimitotic drugs and assembling to microtubules that bind microtubule-associated proteins in the usual way. Electron microscopy confirmed that rehydrated tubulin assembles into normal microtubules that are able to generate motility by interaction with the motor protein kinesin in a cell-free environment. Freeze-drying also preserved preformed microtubules. Rehydrated tubulin and microtubules can be used for preparation of diverse in vitro and in vivo assays as well as for preparation of bionanodevices. Copyright 2009 Elsevier Inc. All rights reserved.
