S. Moreno Díaz de la Espina

Isolation and ultrastructural characterization of the residual nuclear matrix in a plant cell system

We isolated the nuclear matrix of proliferative meristematic root cells of Allium cepa bulbs and characterized it ultrastructurally and by one-dimensional SDS-polyacrylamide gel electrophoresis. The general organization of the nuclear matrix in onion root cells is similar to that found in other eukaryotes extracted under the same conditions. There are three main morphological components: the residual pore-lamina complex, the internal matrix and the residual nucleolus. The matrix has a fibrillo-granular structure composed of 5–10 nm fibrils and 20–25 nm granules. The residual protein matrix has a complex pattern, with several polypeptide bands of 12000 to 70000 Mr. The major bands have Mrs of 62000, 56000, 35000 and 27000.

Ultrastructural Characteristics of Fresh and Frozen-Thawed Ovine Embryos Using Two Cryoprotectants

Abstract Cryopreservation of sheep embryos with ethylene glycol as a protectant appears to be more effective than glycerol, particularly at the morula stage, as has been demonstrated on the basis of in vitro and in vivo development rates after thawing. In this study we compare the ultrastructure of fresh morulae, thawed morulae, and blastocysts cryopreserved with either ethylene glycol or glycerol at the electron microscopic level, to look for cellular damage that could be responsible for proven differences in embryo survival after transfer. Embryos cryopreserved with glycerol showed unequal degrees of conservation even among blastomeres within a single embryo. In morulae, inner blastomeres were completely damaged, whereas external ones appeared to be intact. Both morulae and blastocysts cryopreserved with ethylene glycol showed a higher uniformity in blastomere conservation than embryos with glycerol. The most remarkable features in this experimental group were the presence of desmosomes following tight junctions between blastomeres and the presence of many microvilli on the outer surface of external blastomeres. These characteristics are similar in fresh embryos of the control group. Our results show that ethylene glycol protects membrane and cytoplasmic structures of embryonic cells from cryoinjury much better than glycerol. In vivo survival of embryos confirmed the ultrastructural observations. A limited permeability of glycerol would explain the observed ultrastructural differences in blastomere integrity, which depends on blastomere location and the differences between morulae and blastocysts. We conclude that the low reproductive yield after cryopreservation using glycerol can be attributed to the lack of protection of inner cells.

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.

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.

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 probecontaining 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.

Further investigations on the functional role of two nuclear bodies in onion cells

Summary“Micropuffs” and coiled bodies are two nuclear bodies which have previously been described in a wide range of plant species. In this work, we have demonstrated, by means of ultrastructural cytochemical, autoradiographic and immunocytochemical methods, that the so-called “micropuffs” contain chromatin, which is organized in a supranucleosomal structure, is inactive in transcription, and has no apparent relationship with the nucleolus. These features, as well as the distribution of these structures during the interphase periods, allowed us to conclude that they represent centromeric structures, so, we propose that they be denoted by this name. Coiled bodies show the same silver-staining pattern as the nucleolus organizer (NOR), but do not appear to contain NOR chromatin nor does the early processing of rRNA precursor seem to take place in them. We postulate they are involved in later nuclear RNA processing and storage. This work clarifies some previous misunderstandings with regard to these bodies.

Fine structural organization of a non-reticulate plant cell nucleus

We studied the fine structural organization of the meristematic nucleus in roots of Lycopesicon esculentum (tomato) using ultracytochemical and immunocytochemical approaches. The nucleus has a non-reticulate (i.e. low DNA content) structure whose supramolecular organization differs in some respects from that in reticulate nuclei, principally in the organization of the chromocentres associated with the nuclear envelope, with which centromeric structures appear to be associated. The main difference at the nucleolar level is found in the fibrillar centres, which have a low amount of DNA labelling and in which inclusions of condensed chromatin are present only very rarely. The distribution of nucleolar DNA amongst the nucleolar compartments is similar to that in reticulate nucleoli as demonstrated using an anti-DNA monoclonal antibody. Tomato nuclei have nucleolus-associated bodies or karyosomes, like other plant species with a low DNA content and non-reticulate nuclear organization. The nuclear ribonucleoprotein structures in the inter- and perichromatin regions, namely inter- and perichromatin fibrils and granules, show similar ultrastructural and cytochemical characteristics in both types of nuclei.

Intermediate filament proteins with nuclear functions: NuMA, lamin-like proteins and MFP1

The -helical coiled coil is one of the principal subunit oligomerisation motifs in proteins, consisting of two or more -helices that twist around one another to form a supercoil. Coiled-coil proteins can be classified into two general categories. The first one comprises short coiledcoil domains called leucine zippers that form homoand hetero-dimers, as those found in transcription factors. The second class contains long coiled-coil domains with high polymerisation potential, which form a central rod configuration. The latter constitutes the intermediate filament (IF) protein family. The IF proteins are involved in structural organisation of cells, acting as scaffolds, fixing other proteins to solid state cell components, and also as cytoskeletal motors. They are also involved in molecular recognition and signal transduction (Burkhard et al., 2001). The IF proteins constitute a family of more than 40 different molecular species, classified into six main types (Coulombe et al., 2001); the group V corresponds to the lamins, nuclear proteins that play a role in nuclear organisation and replication (Schirmer et al., 2001). Some IFs associate with centromeres, nuclear matrix (NM), chromatin and the nuclear pore complex, and have DNA binding activity, histone affinity and similarity to NM proteins (Traub and Shoeman, 1994). In contrast to animals, only a reduced number of IFs have been identified in plants. Our previous ultrastructural studies had demonstrated a nucleoskeleton in plants containing both an organised structure underlying the nuclear envelope or lamina (Fig. 1A) (Moreno Diaz De La Espina, 1995) and an internal nucleoskeleton made up from branched knobbed 15–25 nm filaments, with a periodic organisation similar to those of the animal nucleoskeleton, as well as associated aggregates of different sizes (Fig. 1B,C) (Yu and Moreno Diaz De La Espina, 1999). Although from the 1990s we have made some contributions to the identification of IF and coiled-coil proteins in the plant matrix, such as lamins, NuMA and MFP1 (Fig. 1D–F), the molecular composition of the core filaments remains unknown. Nuclear mitotic apparatus protein (NuMA) is a 200– 240 kD coiled-coil protein first identified in animals. NuMA is a prominent component of nuclear core filaments. During mitosis, it relocates to the mitotic spindle and associates with motor proteins, contributing to microtubule stabilisation and spindle organisation. Although its functions during mitosis are well established, studies on its nuclear function are scarce (Taimen et al., 2000). Using an immunological approach, three onion homologues of NuMA, of 210, 220 and 230 kD (Fig. 1D), were identified in the filaments of its nucleoskeleton (Fig. 1G) and in the mitotic spindle (Yu and Moreno Diaz De La Espina, 1999). The potential of NuMA oligomers to form higher order structures in animals (Harborth et al., 1999) and the localisation of AcNuMA in core filaments, suggest its structural role in plant nuclear organisation. Lamin-like proteins with conserved Mr and pI values, antigenically related to animal A and B lamins, were detected in the lamina and the internal NM of several plants (Fig. 1E,H) (see Moreno Diaz De La Espina, 1995 for a review). In spite of all their similarities and of the important nuclear functions played by lamins (Schirmer et al., 2001), lamin genes have not been identified in plants as yet, suggesting that they could have distinctly related nucleoskeletal proteins that functionally replace lamins. Recently, Gindullis et al. (2002) reported a novel family of long coiled-coil proteins from plants, which represents a good candidate for the abovementioned fact. * Corresponding author. Tel.: +34-91-561-1800; fax: +34-91-562-7518. E-mail address: smoreno@cib.csic.es (S. Moreno Diaz De La Espina). Cell Biology International 27 (2003) 233–235 Cell Biology International

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

Summary.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 Mr 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.

G2 checkpoint targets late replicating DNA.

In the multinucleate cells induced in Allium cepa L. meristems, the nuclei surrounded by the largest cytoplasm environment complete replication earlier (advanced nuclei), but have a longer G2, than the others (delayed nuclei). Thus, all nuclei break down the nuclear envelope and start metaphase simultaneously. The present report shows that this synchronization relies on a checkpoint mechanism. When completion of replication was prevented in the delayed nuclei (due to in vivo 5‐aminouracil feeding initiated when the advanced nuclei were already in G2), the metaphase was also further delayed in the advanced ones. In turn, some of the delayed nuclei overrode the G2 checkpoint (adaptation) and entered into mitosis with broken chromatids (Del Campo et al., 1997). Anoxic UVA (313 nm) irradiation apparently prevents the binding of regulatory proteins to Br‐DNA. The present report shows that late replicating sequences are the targets of the checkpoint signal produced by the still replicating nuclei. This signal delays metaphase in the advanced nuclei, whose DNA is already fully replicated. Thus, when the already replicated sequences of late replicating DNA was modified in the advanced nuclei by bromosubstitution followed by anoxic UVA irradiation, they entered into mitosis without any delay, ignoring the inhibitory signals produced by the still replicating nuclei.