Proteins play a major role in the three-dimensional organization of nuclear genome and its function. While histones arrange DNA into a nucleosome fiber, other proteins contribute to higher-order chromatin structures in interphase nuclei, and mitotic/meiotic chromosomes. Despite the key role of proteins in maintaining genome integrity and transferring hereditary information to daughter cells and progenies, the knowledge about their function remains fragmentary. This is particularly true for the proteins of condensed chromosomes and, in particular, chromosomes of plants. Here, we purified barley mitotic metaphase chromosomes by a flow cytometric sorting and characterized their proteins. Peptides from tryptic protein digests were fractionated either on a cation exchanger or reversed-phase microgradient system before liquid chromatography coupled to tandem mass spectrometry. Chromosomal proteins comprising almost 900 identifications were classified based on a combination of software prediction, available database localization information, sequence homology, and domain representation. A biological context evaluation indicated the presence of several groups of abundant proteins including histones, topoisomerase 2, POLYMERASE 2, condensin subunits, and many proteins with chromatin-related functions. Proteins involved in processes related to DNA replication, transcription, and repair as well as nucleolar proteins were found. We have experimentally validated the presence of FIBRILLARIN 1, one of the nucleolar proteins, on metaphase chromosomes, suggesting that plant chromosomes are coated with proteins during mitosis, similar to those of human and animals. These results improve significantly the knowledge of plant chromosomal proteins and provide a basis for their functional characterization and comparative phylogenetic analyses.
- Klíčová slova
- FIBRILLARIN 1, barley, chromatin, flow cytometric sorting, mass spectrometry, mitotic chromosome, perichromosomal layer, protein prediction,
- Publikační typ
- časopisecké články MeSH
The astonishing survival abilities of Vicia faba, one the earliest domesticated plants, are associated, among other things, to the highly effective replication stress response system which ensures smooth cell division and proper preservation of genomic information. The most crucial pathway here seems to be the ataxia telangiectasia-mutated kinase (ATM)/ataxia telangiectasia and Rad3-related kinase (ATR)-dependent replication stress response mechanism, also present in humans. In this article, we attempted to take an in-depth look at the dynamics of regeneration from the effects of replication inhibition and cell cycle checkpoint overriding causing premature chromosome condensation (PCC) in terms of DNA damage repair and changes in replication dynamics. We were able to distinguish a unique behavior of replication factors at the very start of the regeneration process in the PCC-induced cells. We extended the experiment and decided to profile the changes in replication on the level of a single replication cluster of heterochromatin (both alone and with regard to its position in the nucleus), including the mathematical profiling of the size, activity and shape. The results obtained during these experiments led us to the conclusion that even "chaotic" events are dealt with in a proper degree of order.
- Klíčová slova
- 5-ethynyl-2′-deoxyuridine, DNA damage, DNA repair, DNA replication, caffeine, heterochromatin, hydroxyurea, nuclei sorting, premature chromosome condensation, replication stress,
- MeSH
- chromozomy rostlin genetika MeSH
- fluorescence MeSH
- fyziologický stres * MeSH
- heterochromatin metabolismus MeSH
- kinetika MeSH
- meristém fyziologie MeSH
- oprava DNA * MeSH
- poškození DNA MeSH
- regenerace fyziologie MeSH
- replikace DNA * MeSH
- Vicia faba fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- heterochromatin MeSH
TPX2 (Targeting Protein for Xklp2) is an evolutionary conserved microtubule-associated protein important for microtubule nucleation and mitotic spindle assembly. The protein was described as an activator of the mitotic kinase Aurora A in humans and the Arabidopsis AURORA1 (AUR1) kinase. In contrast to animal genomes that encode only one TPX2 gene, higher plant genomes encode a family with several TPX2-LIKE gene members (TPXL). TPXL genes of Arabidopsis can be divided into two groups. Group A proteins (TPXL2, 3, 4, and 8) contain Aurora binding and TPX2_importin domains, while group B proteins (TPXL1, 5, 6, and 7) harbor an Xklp2 domain. Canonical TPX2 contains all the above-mentioned domains. We confirmed using in vitro kinase assays that the group A proteins contain a functional Aurora kinase binding domain. Transient expression of Arabidopsis TPX2-like proteins in Nicotiana benthamiana revealed preferential localization to microtubules and nuclei. Co-expression of AUR1 together with TPX2-like proteins changed the localization of AUR1, indicating that these proteins serve as targeting factors for Aurora kinases. Taken together, we visualize the various localizations of the TPX2-LIKE family in Arabidopsis as a proxy to their functional divergence and provide evidence of their role in the targeted regulation of AUR1 kinase activity.
- Klíčová slova
- Arabidopsis thaliana, aurora kinase 1, in vivo co-localization, kinase assay, phylogeny, targeting protein for Xklp2,
- MeSH
- Arabidopsis cytologie genetika metabolismus MeSH
- kinasy aurora metabolismus MeSH
- mikrotubuly metabolismus MeSH
- proteinové domény MeSH
- proteiny asociované s mikrotubuly analýza genetika metabolismus MeSH
- proteiny huseníčku analýza genetika metabolismus MeSH
- rostlinné geny MeSH
- sekvence aminokyselin MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- kinasy aurora MeSH
- proteiny asociované s mikrotubuly MeSH
- proteiny huseníčku MeSH
- TPX2 protein, Arabidopsis MeSH Prohlížeč
In the plant nucleus, the majority of cellular DNA content is stored and maintained. This makes this highly specialized organelle the major coordinator of almost all essential processes in plant cells such as transcription, DNA replication, and repair. None of these biological pathways can be fully understood without a comprehensive characterization of nuclear proteins. Nevertheless, the interest of the proteomic community in the plant nuclear proteome has been very limited so far. This is probably due to the high integrity of plant cell, presence of many interfering metabolites, and considerable endogenous proteolytic activity which make the sample preparation problematic. Hereby, we describe a novel protocol for the high-throughput plant nuclear protein identification that combines a flow cytometric sorting of formaldehyde-fixed nuclei with protein and peptide separation and their subsequent LC-MS/MS analysis.
- Klíčová slova
- Cell cycle, Flow cytometry, Gel electrophoresis, In-gel digestion, Mass spectrometry, Nuclear proteome, Plant nucleus, Protein analysis,
- MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- jaderné proteiny analýza MeSH
- ječmen (rod) cytologie metabolismus MeSH
- plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
- proteomika metody MeSH
- průtoková cytometrie MeSH
- rostlinné proteiny analýza MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- jaderné proteiny MeSH
- rostlinné proteiny MeSH
Proteins are the most abundant component of the cell nucleus, where they perform a plethora of functions, including the assembly of long DNA molecules into condensed chromatin, DNA replication and repair, regulation of gene expression, synthesis of RNA molecules and their modification. Proteins are important components of nuclear bodies and are involved in the maintenance of the nuclear architecture, transport across the nuclear envelope and cell division. Given their importance, the current poor knowledge of plant nuclear proteins and their dynamics during the cell's life and division is striking. Several factors hamper the analysis of the plant nuclear proteome, but the most critical seems to be the contamination of nuclei by cytosolic material during their isolation. With the availability of an efficient protocol for the purification of plant nuclei, based on flow cytometric sorting, contamination by cytoplasmic remnants can be minimized. Moreover, flow cytometry allows the separation of nuclei in different stages of the cell cycle (G1, S, and G2). This strategy has led to the identification of large number of nuclear proteins from barley (Hordeum vulgare), thus triggering the creation of a dedicated database called UNcleProt, http://barley.gambrinus.ueb.cas.cz/ .
- Klíčová slova
- barley, cell cycle, database, flow-cytometry, localization, mass spectrometry, nuclear proteome, nucleus,
- MeSH
- buněčný cyklus * MeSH
- data mining MeSH
- databáze proteinů * MeSH
- jaderné proteiny klasifikace metabolismus MeSH
- ječmen (rod) cytologie MeSH
- rostlinné proteiny klasifikace metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- jaderné proteiny MeSH
- rostlinné proteiny MeSH
In many plant species, somatic cell differentiation is accompanied by endoreduplication, a process during which cells undergo one or more rounds of DNA replication cycles in the absence of mitosis, resulting in nuclei with multiples of 2C DNA amounts (4C, 8C, 16C, etc.). In some orchids, a disproportionate increase in nuclear DNA contents has been observed, where successive endoreduplication cycles result in DNA amounts 2C + P, 2C + 3P, 2C + 7P, etc., where P is the DNA content of the replicated part of the 2C nuclear genome. This unique phenomenon was termed "progressively partial endoreplication" (PPE). We investigated processes behind the PPE in Ludisia discolor using flow cytometry (FCM) and Illumina sequencing. In particular, we wanted to determine whether chromatin elimination or incomplete genome duplication was involved, and to identify types of DNA sequences that were affected. Cell cycle analysis of root tip cell nuclei pulse-labeled with EdU revealed two cell cycles, one ending above the population of nuclei with 2C + P content, and the other with a typical "horseshoe" pattern of S-phase nuclei ranging from 2C to 4C DNA contents. The process leading to nuclei with 2C + P amounts therefore involves incomplete genome replication. Subsequent Illumina sequencing of flow-sorted 2C and 2C + P nuclei showed that all types of repetitive DNA sequences were affected during PPE; a complete elimination of any specific type of repetitive DNA was not observed. We hypothesize that PPE is part of a highly controlled transition mechanism from proliferation phase to differentiation phase of plant tissue development.
- Klíčová slova
- DNA replication, EdU, Ludisia discolor, cell cycle, endoreduplication, orchids,
- MeSH
- buněčné jádro genetika MeSH
- endoreduplikace genetika MeSH
- genom rostlinný MeSH
- listy rostlin genetika MeSH
- mitóza genetika MeSH
- Orchidaceae genetika MeSH
- polyploidie MeSH
- průtoková cytometrie metody MeSH
- replikace DNA genetika MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Publikační typ
- časopisecké články MeSH
Nuclear proteins are a vital component of eukaryotic cell nuclei and have a profound effect on the way in which genetic information is stored, expressed, replicated, repaired, and transmitted to daughter cells and progeny. Because of the plethora of functions, nuclear proteins represent the most abundant components of cell nuclei in all eukaryotes. However, while the plant genome is well understood at the DNA level, information on plant nuclear proteins remains scarce, perhaps with the exception of histones and a few other proteins. This lack of knowledge hampers efforts to understand how the plant genome is organized in the nucleus and how it functions. This review focuses on the current state of the art of the analysis of the plant nuclear proteome. Previous proteome studies have generally been designed to search for proteins involved in plant response to various forms of stress or to identify rather a modest number of proteins. Thus, there is a need for more comprehensive and systematic studies of proteins in the nuclei obtained at individual phases of the cell cycle, or isolated from various tissue types and stages of cell and tissue differentiation. All this in combination with protein structure, predicted function, and physical localization in 3D nuclear space could provide much needed progress in our understanding of the plant nuclear proteome and its role in plant genome organization and function.
- Klíčová slova
- Cell nucleus, chromatin, genome function, nuclear proteins, plants, proteomics.,
- MeSH
- buněčné jádro metabolismus MeSH
- jaderné proteiny genetika metabolismus MeSH
- proteom genetika metabolismus MeSH
- rostlinné proteiny genetika metabolismus MeSH
- rostliny genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Názvy látek
- jaderné proteiny MeSH
- proteom MeSH
- rostlinné proteiny MeSH
Many proteins are present in the nucleus; some are involved with its structural and functional organization, some with gene expression, and some with cell division. The plant nuclear proteome has not been well explored. Its characterization requires extraction methods which minimize both the artifactual alteration of the proteins and the extent of contamination with non-nuclear proteins. The conventional multi-step fractionation procedure is both laborious and prone to contamination. Here, we describe a single-step method based on flow sorting. The method allows the separation of G1, S and G2 phase nuclei and minimizes the risk of contamination by non-nuclear proteins. Preliminary results obtained using G1 phase cell nuclei from barley root tips indicate that flow sorting coupled with a protein/peptide separation and mass spectrometry will permit a comprehensive characterization of the plant nuclear proteome.
- MeSH
- buněčné jádro genetika MeSH
- interfáze genetika MeSH
- ječmen (rod) genetika MeSH
- proteom genetika MeSH
- proteomika metody MeSH
- průtoková cytometrie metody MeSH
- rostlinné proteiny genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- proteom MeSH
- rostlinné 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
Nitrilases are highly conserved proteins with catabolic activity but much less understood functions in cell division and apoptosis. To elucidate the biological functions of Arabidopsis NITRILASE1, we characterized its molecular forms, cellular localization and involvement in cell proliferation and plant development. We performed biochemical and mass spectrometry analyses of NITRILASE1 complexes, electron microscopy of nitrilase polymers, imaging of developmental and cellular distribution, silencing and overexpression of nitrilases to study their functions. We found that NITRILASE1 has an intrinsic ability to form filaments. GFP-NITRILASE1 was abundant in proliferating cells, distributed in cytoplasm, in the perinuclear area and associated with microtubules. As cells exited proliferation and entered differentiation, GFP-NITRILASE1 became predominantly nuclear. Nitrilase silencing dose-dependently compromised plant growth, led to loss of tissue organization and sustained proliferation. Cytokinesis was frequently aborted, leading to enlarged polyploid cells. In reverse, independently transformed cell lines overexpressing GFP-NITRILASE1 showed slow growth and increased rate of programmed cell death. Altogether, our data suggest that NITRILASE1 homologues regulate the exit from cell cycle and entry into differentiation and simultaneously are required for cytokinesis. These functions are essential to maintain normal ploidy, genome stability and tissue organization.
- MeSH
- aminohydrolasy chemie genetika metabolismus ultrastruktura MeSH
- Arabidopsis cytologie genetika růst a vývoj MeSH
- buněčná diferenciace genetika MeSH
- buněčná smrt genetika MeSH
- buněčný cyklus genetika MeSH
- cytoplazma metabolismus MeSH
- cytoskelet genetika metabolismus MeSH
- hydrolasy působící na anhydridy kyselin genetika MeSH
- nádorové proteiny genetika MeSH
- nestabilita genomu * MeSH
- proliferace buněk MeSH
- regulace genové exprese u rostlin MeSH
- RNA interference MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- aminohydrolasy MeSH
- fragile histidine triad protein MeSH Prohlížeč
- hydrolasy působící na anhydridy kyselin MeSH
- nádorové proteiny MeSH
- nitrilase MeSH Prohlížeč
