Subnuclear compartmentalization and function of actin and nuclear myosin I in plants.

Actins are highly conserved proteins that serve as the basic building blocks of cytoskeletal microfilaments. In animal cells, specific nuclear actin adopts unconventional conformations that are involved in multiple nuclear functions and that associate with nuclear actin binding proteins. However, there is practically no information available about nuclear actin in plants. Indeed, actin has not been detected in the nuclear proteomes of many plants, and orthologs of the main structural nuclear actin-binding proteins have yet to be identified. Here, we have investigated the characteristics, intranuclear compartmentalization, and function of actin in isolated Allium cepa nuclei as well as that of its motor protein nuclear myosin I (NMI). Using conformation-specific antibodies for nuclear actin isoforms, ss-actin, and NMI, the distribution of these proteins was studied in Western blots and by immunocytochemistry. Moreover, the participation of nuclear actin in transcription was analyzed in run on in situ assays and inhibition of RNA polymerases I and II. We show that actin isoforms with distinct solubilities are present in onion nuclei with a consistent subnuclear compartmentalization. Actin and NMI are highly enriched in foci that are similar to transcription foci, although actin is also distributed diffusely in the nucleus and nucleolus as well as accumulating in a subset of the Cajal bodies. Immunogold labeling identified both proteins in the nuclear transcription subdomains and in other subnuclear compartments. In addition, actin and NMI were diffusely distributed in the nuclear matrix.

Characterization, expression and subcellular distribution of a novel MFP1 (matrix attachment region-binding filament-like protein 1) in onion.

MFP1 (matrix attachment region-binding filament-like protein 1) is a conserved nuclear and chloroplast DNA-binding protein encoded by a nuclear gene, well characterized in dicot species. In monocots, only a 90 kDa MFP1-related protein had been characterized in the nucleus and nuclear matrix of Allium cepa proliferating cells. We report here a novel MFP1-related nuclear protein of 80 kDa in A. cepa roots, with M(r) and pI values similar to those of MFP1 proteins in dicot species, and which also displays a dual location, in the nucleus and chloroplasts of leaf cells. However, this novel protein is not a nuclear matrix component. It shows a spotted intranuclear distribution in small foci differing from the nuclear bodies containing the 90 kDa protein. In electron microscopy analysis, the intranuclear foci containing the 80 kDa MFP1 appeared as small loose structures at the periphery of condensed chromatin patches. This protein was also located in the nucleolus. It was abundant in meristematic cells, but its level fell when proliferation stopped. This different expression and distribution, and its preferential location at the boundaries between heterochromatin and euchromatin, suggest that the novel 80 kDa protein might be associated with decondensed DNA and could play a role in chromatin organization.

Telomeric DNA localization on dinoflagellate chromosomes: structural and evolutionary implications.

Dinoflagellates are eukaryotic microalgae with distinct chromosomes throughout the cell cycle which lack histones and nucleosomes. The molecular organization of these chromosomes is still poorly understood. We have analysed the presence of telomeres in two evolutionarily distant and heterogeneous dinoflagellate species (Prorocentrum micans and Amphidinium carterae) by FISH with a probe containing the Arabidopsis consensus telomeric sequence. Telomere structures were identified at the chromosome ends of both species during interphase and mitosis and were frequently associated with the nuclear envelope. These results identify for the first time telomere structures in dinoflagellate chromosomes, which are formed in the absence of histones. The presence of telomeres supports the linear nature of dinoflagellate chromosomes.

Genome remodelling in three modern S. officinarumxS. spontaneum sugarcane cultivars.

This study provides evidence that nuclear and chromosome remodelling has taken place in sugarcane, a vegetative crop with a complex genome derived from interspecific hybridizations between Saccharum officinarum and S. spontaneum. Detailed knowledge on the chromosomal compositions of the three clones analysed was acquired. (1) All hybrid cultivars were found to be aneuploid, affecting both parental genomes (having chromosomes in addition to full genomes), with chromosome numbers from 2n=102-106 in My5514 and up to 2n=113-117 in C236-51. (2) Comparative in situ hybridization showed that about 16% of these chromosomes are inherited from S. spontaneum and less than 5% are recombinant or translocated chromosomes containing sequences of both S. officinarum and S. spontaneum. (3) Differences between the observed DNA contents (estimated by flow cytometry) and those expected from the number of chromosomes, allowed the introgression of additional S. spontaneum or S. officinarum DNA pieces into the B42231 and C236-51 cultivars to be estimated. (4) Size heterogeneity between S. officinarum homologous chromosomes carrying the 18S-5.8S-25S and 5S ribosomal genes (identified by FISH with pTa71 and pTa794, respectively) confirms remodelling occurred by chromosomal interchange events, at least in these homologous chromosomes. (5) Simultaneous visualization of nucleoli and NORs showed that all 18S-5.8S-25S loci were potentially functional in the three clones, independent of their origin and size.

Behaviour of ribosomal genes and nucleolar domains during activation in sugarcane (Saccharum officinarum L.) root primordia: from the unsoaked quiescent state to the steady state of proliferation.

Changes in the organisation of ribosomal genes and nucleolar protein components were analysed in sugarcane (Saccharum officinarum L. cv Cristalina) from the time the quiescent primordia of the radical bands of nodes were stimulated to proliferate by water imbibition, until the meristematic population reached the steady state of proliferation in the growing roots. The kinetics of proliferation was evaluated by flow cytometry, and by the mitotic indexes, in roots of different lengths. All the quiescent cells were in a pre-replicative state (G0), with a 2C DNA content. During their activation process, they progressively reached the steady state of proliferation (mitotic index 7%), with rather fixed frequencies for cells with 2C (G1), 4C (G2), and values between them corresponding to cells replicating their DNA. Decondensation of the ribosomal genes was followed by FISH with probes for the major 25S and 18S rRNAs, and variations in the numbers of nucleoli were recorded in squashed cells after silver staining. The ultrastructure of nucleoli was analysed by electron microscopy, using the EDTA regressive staining for ribonucleoproteins. Quiescent nucleoli showed a clear segregation of their main components: Fibrillar Centre, Dense Fibrillar Component and Cajal's bodies while lacked any Granular Component. However the proliferating ones showed them highly intermingled, except for the Cajal's bodies. Our results revealed a high plasticity of the nucleolar domains in response to cell activation, and allowed to establish a correlation between dispersion of NORs with formation of small fibrillar centers and a nucleolus with all its domains intermingled, and the activation of cell proliferation during root sprouting.

Coiled bodies in nuclei from plant cells evolving from dormancy to proliferation.

To characterise the coiled bodies in meristematic nuclei of Saccharum officinarum, immunofluorescence labelling with antibodies against components of the splicing (U2B'' and Sm core protein B) and pre-rRNA processing (fibrillarin) complexes was used in cells from the dormant root primordia and from roots at different times after activation to the steady state of proliferation. The number, size and distribution of coiled bodies varied in the meristematic tissue depending on cell activity. While G0 cells in the dry primordia and proliferating cells showed a similar number of coiled bodies attached to their nucleoli, the number of nucleoplasmic coiled bodies greatly increased after the primordia were stimulated to proliferate. Their number remained steady from the time the meristematic population reached the steady state of proliferation, as estimated by flow cytometry. Fractionation studies demonstrated that coiled bodies are a part of the underlying nuclear matrix. Comparison of immunocytochemical and cytochemical data from confocal and electron microscopical studies demonstrated that the nucleolar and nucleoplasmic coiled bodies detected by confocal microscopy shared many features, suggesting that they form a family of closely related structures.

Characterisation and high-resolution distribution of a matrix attachment region-binding protein (MFP1) in proliferating cells of onion.

The first matrix attachment region (MAR)-binding protein sequenced in plants, MFP1, has been characterised in two dicot species. Based on their antigenic relationship, we report here the conservation of MFP1-like proteins in proliferating root cells of onion (Allium cepa L). Two MFP1-like proteins with different molecular masses and solubilities were detected. The most abundant was a 90-kDa basic protein, presenting several separate spots in two-dimensional blots. The MFP1 was partially soluble and, similar to the proliferating cell nuclear antigen (PCNA)-labelled replication factories in the nucleus and nuclear matrix, was localised at discrete foci as detected by confocal microscopy. High-resolution immunolocalisation of MFP1 by electron microscopy identified the foci as nuclear structures, some of them containing PCNA, which are ultrastructurally similar to the replication factories described in animal cells. Our data provide the first report on MFP1-like proteins in the Alliaceae. In addition, we present evidence of the presence of AcMFP1 in the putative replication factories.

The plant nucleoskeleton: ultrastructural organization and identification of NuMA homologues in the nuclear matrix and mitotic spindle of plant cells.

In the present work we investigate the structural organization of the nucleoskeleton of Allium cepa meristematic root cells. Resinless sections reveal for the first time a residual filamentous network in plant nuclei. This network is composed of branched knobbed filaments with associated globular structures, connected to the lamina and to the dense aggregates of different sizes. Results of immunoblotting show that many components of this network are homologues of intermediate filament-type proteins. NuMA, a coiled-coil protein related to intermediate filaments, found in animal cells, can also be detected in this plant nuclear matrix system. Immunofluorescence reveals a diffuse distribution of the animal NuMA homologues in plant nuclear core filaments in interphase. Resinless immunoelectron microscopy further reveals a distribution along the extended filaments and the dense aggregates. During mitosis, in contrast to the accumulation at the poles in animal cells, NuMA homologues in plant onion cells show a diffuse pattern, which may correspond to the spindle matrix. Our data are the first report of the conservation in plants of NuMA proteins, which may be involved in both nuclear and mitotic spindle organizations.

Post-mitotic assembly of the nucleolus. I. The internal matrix network is a recruitment site for processing nucleolar components in prenucleolar bodies.

The aim of this work was to investigate whether the nuclear matrix could provide the nucleation sites for dispersed parental nucleolar components to form post-mitotic prenucleolar bodies (PNBs). For this purpose, nuclear matrices from asynchronous populations of onion cells were fractionated, and the distribution of the insoluble components of the nucleolar processing complexes in the matrices were analysed by fibrillarin immunolabelling. The ultra-structural organization of the nuclear matrix of cells from late telophase to late G1, corresponding to the period of nucleolar reassembly and activation, was also analysed. Our results demonstrate that PNBs are structural components of the telophasic nuclear matrix and that this structure provides recruitment and assembly sites for the components of the nucleolar processing machinery, and suggests that the telophasic matrix network is involved in the early steps of post-mitotic nucleologenesis.

In situ localization of nucleolin in the plant nucleolar matrix.

The analysis of isolated nucleolar matrices from onion cells by light and electron microscopy, 2-D separation of proteins, and confocal microscopy has confirmed the existence of an organized nucleolar matrix with a complex protein composition to which are attached the insoluble processing complexes. In the present work, we present evidence from immunoblotting, immunofluorescence, immunogold labeling, and preferential cytochemical staining with bismuth salts that an insoluble fraction of the multifunctional protein nucleolin, is a component of the onion nucleolar matrix, and analyse its ultrastructural distribution in the described domains of the matrix.

Nuclear matrix isolated from plant cells.

Residual nuclear matrices can be successfully obtained from isolated nuclei of different monocot and dicot plant species using either high ionic or low ionic extraction protocols. The protein composition of isolated nuclear matrices depends on the details of isolation protocols. They are stable and present in all cases, a tripartite organization with a lamina, nucleolar matrix, and internal matrix network, and also maintain some of the basic architectural features of intact nuclei. In situ preparations demonstrate the continuity between the nuclear matrix and the plant cytoskeleton. Two-dimensional separation of isolated plant nuclear matrix proteins reveals a heterogeneous polypeptide composition corresponding rather to a complex multicomponent matrix than to a simple nucleoskeletal structure. Immunological identification of some plant nuclear matrix components such as A and B type lamins, topoisomerase II, and some components of the transcription and splicing machineries, internal intermediate filament proteins, and also specific nucleolar proteins like fibrillarin and nucleolin, which associate to specific matrix domains, establish a model of organization for the plant nuclear matrix similar to that of other eukaryotes. Components of the transcription, processing, and DNA-anchoring complexes are associated with a very stable nucleoskeleton. The plant matrix-attached regions share structural and functional characteristics with those of insects, vertebrates, and yeast, and some of them are active in animal cells. In conclusion, the available data support the view that the plant nuclear matrix is basically similar in animal and plant systems, and has been evolutionarily conserved in eukaryotes.

Dinoflagellates have a eukaryotic nuclear matrix with lamin-like proteins and topoisomerase II.

Unicellular Dinoflagellates represent the only eukaryotic Phylum lacking histones and nucleosomes. To investigate whether Dinoflagellates do have a nuclear matrix that would modulate the supramolecular organization of their non-nucleosomal DNA and chromosomes, cells of the free-living unarmored Dinoflagellate Amphidinium carterae were encapsulated in agarose microbeads and submitted to sequential extraction with non-ionic detergents, nucleases and 2 M NaCl. Our results demonstrate that this species has a residual nuclear matrix similar to that of vertebrates and higher plants. The cytoskeleton-nuclear matrix complex of A. carterae shows a relatively intricate polypeptide pattern. Immunoblots with different antibodies reveal several intermediate filament types of proteins, one of which is immunologically related to vertebrate lamins, confirming that these proteins are ancestral members of the IF family, which is highly conserved in eukaryotes. A topoisomerase II homologue has also been identified in the nuclear matrix, suggesting that these structures could play a role in organizing the Dinoflagellate DNA in loop domains. Taken together our results demonstrate that the nuclear matrix is an early acquisition of the eukaryotic nucleus, independent of histones and nucleosomes in such a way that the mechanisms controlling the two levels of organization in eukaryotic chromatin would be molecularly and evolutionarily independent.

Immunological characterization of lamins in the nuclear matrix of onion cells.

We have used polyclonal and monoclonal antibodies against different lamins from vertebrates, and the IFA antibody recognizing all kinds of intermediate filament proteins, to investigate the lamins of the nuclear matrix of Allium cepa meristematic root cells. All the antibodies react in the onion nuclear matrix with bands in the range of 60-65 kDa, which are enriched in the nuclear matrix after urea extraction, and do not crossreact with other antibodies recognizing intermediate filaments in plants (AFB, anti-vimentin and MAC 322), ruling out crossreaction with contaminating intermediate filaments of cytoplasmic bundles. In 2-D blots the chicken anti-lamin serum reacts with one spot at 65 kDa and pI 6.8 and the anti B-type lamin antibodies with another one at 64 kDa and pI 5.75. Both crossreact with IFA. The lamin is localized at the nuclear periphery and the lamina by indirect immunofluorescence. Immunogold labelling of nuclear matrix sections reveals that the protein is not only associated with the lamina, but also with the internal matrix. Taken together these results reveal that higher plants, which do not possess an organized network of cytoplasmic intermediate filaments, nevertheless present a well-organized lamina containing lamins in which at least one of them is immunologically related to vertebrate lamin B. Our data confirm that lamins are very old members of the intermediate filament proteins that have been better conserved in plants during evolution than their cytoplasmic counterparts.

Ribonucleoprotein components of root meristematic cell nuclei of the tomato characterized by application of mild loosening and immunocytochemistry.

Immunocytochemistry and hypotonic-formaldehyde fixation have been used to study the extranucleolar ribonucleoprotein (RNP) constituents of the nucleus of tomato root meristematic cells. The study of the distribution of small nuclear uridine-rich RNPs (snRNP) by means of a monoclonal anti-Sm antibody recognizing a 29-kDa protein in plants, after standard fixation, shows a preferential labeling of the perichromatin region and a lower labeling of the interchromatin space. These results suggest that in the tomato there is a perichromatin region similar to that of animal cells, in which much of the nonnucleolar transcription and splicing takes place. In hypotonic-formaldehyde-detergent-fixed nuclei, fibrogranular polyparticles have been visualized reacting with anti-snRNP antibody. These structures are frequently associated with filaments of extended chromatin characterized by their reaction with an anti-DNA monoclonal antibody.

Nucleolar fibrillar centres in plant meristematic cells: ultrastructure, cytochemistry and autoradiography.

In plant cells nucleolar fibrillar centres (FCs) undergo ultrastructural changes, depending on the nucleolar activity. We have found two types of FC structure in nucleoli with either high or low activity, to which we have given the conventional names of homogeneous and heterogeneous, respectively. The first type is characterized by the presence of fibres that we describe structurally and cytochemically as decondensed chromatin; the second type, in addition to these fibres, contains a variable number of dense cores made up of condensed chromatin. Moreover, RNA does not appear to be present in FCs, while proteins are a major component. Autoradiography after tritiated uridine incorporation shows that FCs are not the site of transcription, but that this takes place in the fibrillar component; the same result is obtained using the lead acetate fixation technique for detecting orthophosphate ions. This fact leads us to think that FCs are not the whole interphasic counterpart of the mitotic nucleolar organizing region (NOR), as stated by other authors, but only the portion of the NOR that is temporarily inactive in transcription; the transcriptionally active part of the NOR is in the fibrillar component, bound to its earliest product of transcription. Thus, FCs and the fibrillar component constitute a functional unit.

Occurrence of nucleolar material in the cytoplasm of plant cells.

This paper deals with the induction of cytoplasmic nucleolar bodies in meristematic Allium cepa L. cells after treatment with drugs which interfere with nucleolar functionality. The drugs which interfere with protein synthesis failed to produce these bodies. The ultrastructure origin and physiological significance of these bodies are discussed here, as well as their relation with the mitotic prenucleolar bodies (Moreno-Díaz de la Espina et al., 1976).

Alteration of nucleolar dispersion in prophase by 5-FUdR treatment.

The action of 5-Fluorodeoxyuridine (FUdR) used as an inhibitor of RNA synthesis on the nucleolar evolution during mitosis, has been studied in meristematic cells. Under FUdR treatment the nucleolar dispersion appears as a continuous process, but generally it is not completed and nucleolar remnants remain throughout the whole mitosis. The nucleolar material which was dispersed is transported by the mitotic chromosomes, and in telophase contributed to the formation of the new nucleolus. The non-dispersed part persisted in the cytoplasm during telophase, coexisting with both the prenucleolar bodies and the new nucleolus which was being formed. Our results suggest the necessity of some kind of RNA synthesis, preferentially blocked by FUdR, for nucleolar dispersion to take place.