Francisco-Javier Medina

Cytological approach to the nucleolar functions detected by silver staining

The pattern of nucleolar silver staining was determined in the interphase and mitosis of plant cells using two cytological techniques, the selectivity of which was tested densitometrically. For complete accuracy, silver grains were removed and the sections were then observed under uranyl and lead staining. During interphase, the highest density of silver grains occurred in the portions of the dense fibrillar component nearest to the fibrillar centres; the latter were also stained except for the condensed chromatin inclusions typical of the heterogeneous type. During mitosis, not only the nucleolar organiser region (NOR) was stained, but also the prenucleolar material in anaphase and telophase. Two different proteins or sets of proteins are suggested to be responsible for this staining pattern; one of them is associated with NOR chromatin (which is not contained in the mitotic prenucleolar material) and the other with early ribosomal precursors (which have never been found in fibrillar centres). From these two proteins, the “Ag-NOR” reveals either transcriptionally active or potentially active chromatin, characterized by an uncoiled, extended structure, regardless of the state of transcription. This is the structural feature detected by Ag-NOR staining.

Identification and localization of a nucleolin homologue in onion nucleoli

A protein homologous to nucleolin, a major nucleolar protein with multifunctional features involved in pre-rRNA synthesis and early processing, has been identified and localized in situ in onion root meristematic cells by different techniques, which have included the use of an antibody raised against hamster nucleolin. The protein was identified on Western blots of nucleolar proteins as a 64-kDa band, by means of the anti-nucleolin antibody, bismuth staining, and the silver staining-nucleolar organizer (Ag-NOR) method. The experiments also suggested that nucleolin could be a target of these two cytochemical stainings. Although the 64-kDa band corresponds to a major nucleolar protein, it is a minor one among total nuclear proteins. The same techniques were used in situ at the ultrastructural level, and the immunogold detection of the nucleolin homologue was quantitatively evaluated. The protein accumulates in the transition area from nucleolar fibrillar centers to the dense fibrillar component, which is considered to be the structural result of ribosomal gene transcription. Out of this transition area, the dense fibrillar component may be divided into two regions, proximal and distal with respect to fibrillar centers, which show, respectively, the significant and unsignificant presence of nucleolin; we interpret this fact as the expression of the topological arrangement of pre-rRNA processing. Fibrillar centers themselves showed a weak but significant labeling with the anti-nucleolin antibody. However, bismuth staining was absent from the interior of fibrillar centers, indicating that the nucleolin in them is not phosphorylated. Ag-NOR staining uniformly covered fibrillar centers and the dense fibrillar component (at least in its proximal region), but it did not stain condensed chromatin inclusions in heterogeneous fibrillar centers, showing that the binding of nucleolin to chromatin is associated with its decondensation. This work provides additional evidence of the high phylogenetic conservation of molecular motifs which take part in ribosome biogenesis.

NopA64, a novel nucleolar phosphoprotein from proliferating onion cells, sharing immunological determinants with mammalian nucleolin

Abstract. Five major soluble nuclear proteins associated with cell proliferation were identified in Allium cepa L. root cells. One of them, of 64 kDa, was revealed by Western blotting with anti-mammalian nucleolin antibodies. A polyclonal antibody raised against this protein, which we have named NopA64, localised it in the nucleolus as well as in nuclear coiled bodies. Together with NopA64, the antibody also revealed a smaller form, called NopA61. Both proteins were present in the soluble ribonucleoprotein fraction and in the nuclear matrix of proliferating cells, but NopA61 was the only form revealed in differentiated cells. NopA64 contained epitopes also present in other plants, in mammalian nucleolin and in its yeast homologue, gar2. In mammals, the highest homology was with 50-kDa nucleolin fragments containing the RNA-binding motifs and the glycine-arginine-rich (GAR) domain. NopA64 was moderately phosphorylated in vitro by exogenous casein kinase II and cdc2 kinase, whereas NopA61 was highly phosphorylated by casein kinase II. Furthermore, NopA61 was the only band detected after dephosphorylation as well as after endoproteolysis of NopA64. This protein could be one of the various functional homologues of mammalian nucleolin in plant cells.

Subnucleolar location of fibrillarin and NopA64 in Lepidium sativum root meristematic cells is changed in altered gravity.

Summary.Fibrillarin and the plant nucleolin homolog NopA64 are two important nucleolar proteins involved in pre-rRNA processing. In order to determine the effects of the altered gravity environment on the nucleolus, we have investigated the location of fibrillarin and NopA64 in nucleolar subcomponents of cress (Lepidium sativum L.) root meristematic cells grown under clinorotation, which reproduces an important feature of microgravity, namely, the absence of the orienting action of a gravity vector, and compared it to the location in control cells grown in normal 1 g conditions. Prior to these experiments, we report here the characterization of cress fibrillarin as a 41 kDa protein which can be isolated from meristematic cells in three nuclear fractions, namely, the soluble ribonucleoprotein fraction, the chromatin fraction, and the nuclear-matrix fraction. Furthermore, as reported for other species, the location of both fibrillarin and NopA64 in the cress cell nucleolus was in zones known to contain complex ribonucleoprotein particles involved in early pre-rRNA processing, i.e., processomes. Under altered gravity, a decrease in the quantity of both fibrillarin and NopA64 compared to controls was observed in the transition zone between fibrillar centers and the dense fibrillar component, as well as in the bulk of the dense fibrillar component. These data suggest that altered (reduced) gravity results in a lowered level of functional activity in the nucleolus.

Microwave irradiation improvements in the silver staining of the nucleolar organizer (Ag-NOR) technique

The well-know technique of silver staining of the nucleolar organizer (Ag-NOR) is improved in contrast, selectivity and speed when performed with microwave irradiation. The Ag-NOR technique is a very useful tool for studies on the functional morphology and molecular architecture of the nucleolus, and is reputed to be one of the best techniques for diagnosis and prognosis of cancer lesions. To test the generality of the enhancing effects. our study has involved the use of both mammalian and plant cells. Two steps in the process are improved quantitatively by microwave irradiation: fixation and staining itself. Fixation with the ethanol-based reagent, Kryofix, for 3 min in the microwave oven, resulted in good structural preservation at the optical level, and enhanced the contrast and selectivity of silver staining. On the contrary, we found that neither glutaraldehyde fixation, nor a treatment of sections with Carnoys solution, improved Ag-NOR staining. After an analysis of the effects of the different substances involved in sample preparation, we conclude that ethanol is an essential factor for fixation for nucleolar staining, particularly if aldehydes are eliminated from fixative solutions. The process of staining was performed with a drop of staining solution on a semithin section of plastic-embedded tissue intthe microwave oven for 1 min. Staining under these conditions always improved the visualization of nucleoli, regardless of the fixation procedure. Therefore, microwave irradiation at both steps is recommended for giving the best results. Microwave irradiation probably enhances fixation by controlled heat, whereas the increase in reactivity of the staining solution is a direct effect by the microwaves on the silver ions themselves. We used this method to study nucleolar materials during mitosis in proliferating plant cells. Current applications of Ag-NOR staining can be improved with this technical modification.

Mechanisms of disruption of meristematic competence by microgravity in Arabidopsis seedlings

Experiments performed in actively proliferating plant cells both in space and simulated microgravity have evidenced a common effect: cell proliferation appears enhanced whereas cell growth is depleted. Coordination of cell growth and proliferation, called meristematic competence, is a major feature of meristematic cells and its disruption may lead to important alterations in the developmental pattern of the plant. Auxin is known to be a mediator of the transduction of the gravitropic signal and a regulator of the rates of growth and proliferation in meristematic cells, as well as of their further differentiation. Therefore, gravity sensing, gravitropism, auxin levels, and meristematic competence are mutually interrelated. However, our experiments in simulated microgravity, using both mechanical and magnetic levitation technologies, have revealed that this interdependence is neither strict nor univocal and may include additional factors and mechanisms. Available data indicate that altered gravity may affect cell growth and proliferation by mechanisms alternative to the transduction of the gravitropic signal perceived by columella cells in the root tip. These mechanisms would include gravity sensing independent from statolith displacement and transduction mediators other than polar auxin transport.

Microsome-associated proteome modifications of Arabidopsis seedlings grown on board the International Space Station reveal the possible effect on plants of space stresses other than microgravity

Growing plants in space for using them in bioregenerative life support systems during long-term human spaceflights needs improvement of our knowledge in how plants can adapt to space growth conditions. In a previous study performed on board the International Space Station (GENARA A experiment STS-132) we evaluate the global changes that microgravity can exert on the membrane proteome of Arabidopsis seedlings. Here we report additional data from this space experiment, taking advantage of the availability in the EMCS of a centrifuge to evaluate the effects of cues other than microgravity on the relative distribution of membrane proteins. Among the 1484 membrane proteins quantified, 227 proteins displayed no abundance differences between µ g and 1 g in space, while their abundances significantly differed between 1 g in space and 1 g on ground. A majority of these proteins (176) were over-represented in space samples and mainly belong to families corresponding to protein synthesis, degradation, transport, lipid metabolism, or ribosomal proteins. In the remaining set of 51 proteins that were under-represented in membranes, aquaporins and chloroplastic proteins are majority. These sets of proteins clearly appear as indicators of plant physiological processes affected in space by stressful factors others than microgravity.