Lourdes Teresa Agredano-Moreno

Distinctive expression and functional regulation of the maize (Zea mays L.) TOR kinase ortholog

TOR (Target of rapamycin) kinase is a central component of a signal transduction pathway that regulates cellular growth in response to nutrients, mitogens and growth factors in eukaryotes. Knowledge of the TOR pathway in plants is scarce, and reports in agronomical relevant plants are lacking. Previous studies indicate that Arabidopsis thaliana TOR (AtTOR) activity is resistant to rapamycin whereas maize TOR (ZmTOR) is not, suggesting that plants might have different regulation mechanisms for this signal transduction pathway. In the present work maize ZmTOR cDNA was identified and its expression regulation was analyzed during germination on different tissues at various stages of differentiation and by the main ZmTOR regulators. Our results show that ZmTOR contains all functional domains characteristic of metazoan TOR kinase. ZmTOR expression is highly regulated during germination, a critical plant development period, but not on other tissues of contrasting physiological characteristics. Bioinformatic analyses indicated that maize FKBP12 and rapamycin form a functional structure capable of targeting the ZmTOR protein, similar to other non-plant eukaryotes, further supporting its regulation by rapamycin (in contrast with the rapamycin insensitivity of Arabidopsis thaliana) and the conservation of rapamycin regulation through plant evolution.

The distribution of phosphorylated SR proteins and alternative splicing are regulated by RANBP2

SR splicing factors are distributed in the speckled pattern in the nucleus. Alternative pre-mRNA splicing is regulated through nuclear distribution of phosphorylated SR splicing factors, which is specifically regulated by the RANBP2 system in mammalian cell lines, as well as in mouse tissues.

The ultrastructural study of the interphase cell nucleus of Lacandonia schismatica (Lacandoniaceae: Triuridales) reveals a non-typical extranucleolar particle

By light and electron microscope cytochemistry we characterized the interphase nucleus of Lacandonia schismatica, the only known species of the new plant family Lacandoniaceae, whose most peculiar feature is the inverted position of the sexual organs, an aspect never found before among flowering plants. Furthermore, we compare it to Triuris alata, a related species, to Voyria aphylla (a dicotyledon), to Gymnosiphon divaricatus (a monocotyledon) and also to saprophytes. The reticulated chromatin of L schismatica and T alata is similar to that of other monocotyledons. In addition, we describe a unique type of RNP granules in the interchromatin space which are about 32 ± 3 nm SD in diameter and occur as huge clusters. They are intermediate in size and spatial distribution between inter‐ and peri‐chromatin granules. We term them ‘Lacandonia granules’. The granules were also found in T alata. They are 31 ± 2 nm in diameter. No significant differences in size were observed between them (P > 0.05). Synaptonemallike complexes and ring‐shaped structures were seen in interphase nuclei of somatic cells of these species. Coiled and nucleolus‐associated bodies, as well as centromeres were also found in these two organisms. On the contrary, Vaphylla and G divaricatus display a chromocentric nuclear organization. The nuclear similarities between L schismatica and T alata suggest extremely close phylogenetic relationships between them.

New evidence that Lacandonia granules are ultrastructurally related to perichromatin and Balbiani ring granules

Summary— Lacandonia granules are abundant non‐typical extranucleolar ribonucleoprotein particles found in the nucleus of Lacandonia schismatica, a rare plant showing spatial inversion of sex organs. In the present study, changes in the number of Lacandonia granules during flower development, and the presence of SR proteins and poly(A)+ RNA in the nuclei of L. schismatica were analyzed by electron microscopy, immunoelectron microscopy and ultrastructural in situ hybridization. Our results show an important reduction in the number of Lacandonia granules in the nuclei of cells of opened (post‐anthesis) in relation to unopened (pre‐anthesis) flowers, where granules are very abundant. The SR family of splicing factors and poly(A)+ RNA are present in both perichromatin fibers and Lacandonia granules. The developmental behavior, the presence of SR proteins, recently involved in post‐splicing events, poly(A)+ RNA and the reported absence of snRNPs splicing factors in Lacandonia granules, suggest that these particles are involved in postranscriptional events as storage and/or transport of mRNAs. A similar situation is present in other nuclear RNP as perichromatin granules present in mammals and Balbiani ring granules of salivary glands of Chironomus. Based on similarities in morphological, developmental behavior, immunocytochemistry and in situ hybridization results, we conclude that Lacandonia, perichromatin and Balbiani ring granules may be also functionally similar structures.

Cytochemical and immunocytochemical studyof nuclear structures of Lacandonia schismatica

Summary— The interphase nucleus of the cells of several tissues of Lacandonia schismatica was studied using electron microscopy cytochemical and immunocytochemical methods. The EDTA staining procedure, preferential for RNP, contrasted the Lacandonia granules and perichromatin fibrils. These granules were found to be relatively resistant to RNAse hydrolysis, but they were easily digested if RNAse treatment was carried out after a short hydrolysis with pronase. Bismuth oxynitrate stained granular structures about 17 nm in diameter and the periphery of a few Lacandonia granules. The anti=snURNPs bound to RNP‐containing fibrils in the perichromatin and interchromatin space and also to the periphery of some Lacandonia granules. Immunolabeling of DNA demonstrated numerous filaments of extended chromatin in the perichromatin and interchromatin spaces which were closely related to Lacandonia granules. These observations suggested that Lacandonia granules are equivalent to Balbiani ring granules of nuclei with polytene chromosomes and to perichromatin granules of other plant and animal nuclei. The small number of Lacandonia granules labeled in their periphery by anti‐snURNP mAb were interpreted as being immature granules in the process of formation. The external or annular part of the ring‐shaped structures is heavily labeled by anti‐URNP mAbs but scarcely stained by the EDTA procedure. These features indicate that this region contains abundant proteins associated with snURNAs but probably little snURNAs. The synaptonemal‐like complexes previously found in the interphase nuclei of Lacandonia are formed by two parallel masses of compact chromatin, which react with anti‐DNA, and a central clear space crossed by fiber. Most of these fibers do not react with tests for DNA or RNA and are probably composed of proteins. The EDTA procedure contrasts some fibrils at the internal aspect of the masses of compact chromatin, suggesting the presence of remnants of the lateral element of a synaptonemal complex.

Lacandonia granules are present in Ginkgo biloba cell nuclei

Summry— Lacandonia schismatica is a rare flowering plant with the sex organs spatially inverted. Several aspects of its cell biology are now known. Interestingly, within the cell nucleus, the chromatin is reticulated and it is associated to a novel structure named Lacandonia granules, a very abundant ribonucleoprotein particle showing similarities to perichromatin and Balbiani ring granules, which are involved in nuclear mRNA metabolism. To see whether these particles are present in other plants, we study the nucleus of Ginkgo biloba, a non‐flowering plant. Light, electron and atomic force microscopy show that the cell nuclei of G. biloba are reticulated. Ultrastructural analysis showed that in the nucleoplasm, abundant intranuclear particles 32 nm in diameter are present. The EDTA regressive staining suggested that they contain RNA. Ultrastructural in situ hybridization confirmed the presence of RNA in these particles. Therefore, we conclude that the nuclei of G. biloba are reticulated and contain Lacandonia granules. We suggest that these particles may also be present in other plants.

Status epilepticus triggers early mitochondrial fusion in the rat hippocampus in a lithium-pilocarpine model

Many reports investigating the hippocampus have demonstrated an increase in neuronal damage, cellular loss, oxidative stress and mitochondrial DNA damage during status epilepticus (SE); however, information regarding alterations in mitochondrial fission and fusion events in SE is lacking. The aim of the present study was to examine the possible imbalance between mitochondrial fission and fusion in the hippocampus of male rats after acute seizure mediated by SE. In this study, we used ninety animals were randomly divided into control and SE groups and subjected to the lithium-pilocarpine model of epilepsy. Hippocampi were obtained at 3, 24 and 72h after SE, and the cytoplasmic and mitochondrial fractions of the cells were used to analyze changes in the Drp1 and Fis1 fission proteins and the Mfn1 and Opa1 fusion proteins by western blot analysis. Moreover, changes in the expression of fission and fusion mRNA transcripts were evaluated by real-time PCR. Mitochondrial morphology was also analyzed using standard transmission electron microscopy. Our data showed that the fission-related mRNA Drp1 was down-regulated rapidly after SE, while Fis1 did not show any significant changes in expression. Moreover, the mitochondrial fusion-associated proteins Mfn1 and Opa1 exhibited an increase in expression at 72h after SE. Electron microphotography revealed several morphological changes, such as swollen mitochondria and damage of the inner mitochondrial membrane, at 24h; at 72h elongation of some mitochondrial was also observed. Our results suggest that after the initiation of SE, the main regulator of the fission mRNA Drp1 is down-regulated, which in turn regulates mitochondrial fission and leads to an increase in the Mfn1 and Opa1 proteins to induce mitochondrial fusion, suggesting an imbalance of the fission and fusion processes.

The cell wall DUF642 At2g41800 (TEB) protein is involved in hypocotyl cell elongation

In plants, the cell wall is a complex and dynamic structure comprising high molecular weight carbohydrates and proteins. The cell wall plays an important role in several stages of the plant life cycle, including cell division, elongation and differentiation. The DUF642 family of cell wall proteins is highly conserved in spermatophytes and might be involved in pectin structural modifications. Particularly, At2g41800 is one of the most highly induced genes during the M/G1 phases of the cell cycle, and the protein encodes by this gene has been detected in cell wall proteomes of cell suspension cultures. In the present study, the expression of At2g41800 (TEB) was confirmed in primary and lateral roots, stigmatic papillae and hypocotyls. Subcellular localization studies showed that TEB is located in the cell wall. The root length and lateral root density were not affected in either of the two teb mutants studied, but the length of the hypocotyls from seedlings grown under light and dark conditions was increased. Immunogold labelling studies using JIM5 antibodies on sections of hypocotyl epidermal cells showed an important reduction of gold particles in teb mutants. The results suggested that TEB is involved in hypocotyl elongation.

Visualization of internal in situ cell structure by atomic force microscopy

Light and electron microscopy have been used to study cell structure for many years, but atomic force microscopy is a more recent technique used to analyze cells, mainly due to the absence of techniques to prepare the samples. Isolated molecules or organelles, whole cells, and to a lesser extent in situ cell structure have been observed by different atomic force microscopy imaging modes. Here, we review efforts intended to analyze in situ the cell structures using approaches involving imaging of the surface of semithin sections of samples embedded in resin and sections prepared with an ultramicrotome. The results of such studies are discussed in relation to their implications to analyze the fine structure of organelles at the nanoscale in situ at enhanced resolution compared to light microscopy.

Ultrastructural and physiological changes induced by different stress conditions on the human parasite Trypanosoma cruzi.

Trypanosoma cruzi is the etiological agent of Chagas disease. The life cycle of this protozoan parasite is digenetic because it alternates its different developmental forms through two hosts, a vector insect and a vertebrate host. As a result, the parasites are exposed to sudden and drastic environmental changes causing cellular stress. The stress response to some types of stress has been studied in T. cruzi, mainly at the molecular level; however, data about ultrastructure and physiological state of the cells in stress conditions are scarce or null. In this work, we analyzed the morphological, ultrastructural, and physiological changes produced on T. cruzi epimastigotes when they were exposed to acid, nutritional, heat, and oxidative stress. Clear morphological changes were observed, but the physiological conditions varied depending on the type of stress. The maintenance of the physiological state was severely affected by heat shock, acidic, nutritional, and oxidative stress. According to the surprising observed growth recovery after damage by stress alterations, different adaptations from the parasite to these harsh conditions were suggested. Particular cellular death pathways are discussed.