Zdeněk Opatrný

Comparison of Several Digital and Stereological Methods for Estimating Surface Area and Volume of Cells Studied by Confocal Microscopy

BACKGROUND The implementation of different methods for estimating the surface area and volume of cells studied by confocal microscopy was developed. The methods were compared from the point of view of their precision, applicability and efficiency. METHODS Interactive stereological methods (spatial grid method, fakir method, Cavalieri principle) as well as automatic digital methods (digital Crofton method, voxel counting, triangulation method, iso-intensity contouring method) were considered. The methods were tested on model geometrical solids and on real volume images consisting of a stack of serial sections encompassing entire tobacco BY-2 cells or cell chains. RESULTS It is shown that many of the studied methods are very precise when applied to cells of simple or moderately complex shapes. The automatic digital methods are fast and precise but their applicability is limited by the necessity to segment automatically the object surface and to find an optimal resolution. This limitation is not present in stereological methods which are applied interactively and thus are more time-consuming. CONCLUSIONS The presented implementations of the fakir method and the Cavalieri principle enable interactive, unbiased and efficient estimation of the cell surface area and volume. The recommended steps for measuring the surface area and/or volume of objects studied by confocal microscopy are described.

The role of actin isoforms in somatic embryogenesis in Norway spruce

BackgroundSomatic embryogenesis in spruce is a process of high importance for biotechnology, yet it comprises of orchestrated series of events whose cellular and molecular details are not well understood. In this study, we examined the role of actin cytoskeleton during somatic embryogenesis in Norway spruce line AFO 541 by means of anti-actin drugs.ResultsApplication of low doses (50-100 nM) of latrunculin B (Lat B) during the maturation of somatic embryos predominantly killed suspensor cells while leaving the cells in meristematic centres alive, indicating differential sensitivity of actin in the two cell types. The treatment resulted in faster development of more advanced embryos into mature somatic embryos and elimination of insufficiently developed ones. In searching for the cause of the differential actin sensitivity of the two cell types, we analysed the composition of actin isoforms in the culture and isolated four spruce actin genes. Analysis of their expression during embryo maturation revealed that one actin isoform was expressed constitutively in both cell types, whereas three actin isoforms were expressed predominantly in suspensor cells and their expression declined during the maturation. The expression decline was greatly enhanced by Lat B treatment. Sequence analysis revealed amino-acid substitutions in the Lat B-binding site in one of the suspensor-specific actin isoforms, which may result in a different binding affinity for Lat B.ConclusionsWe show that manipulating actin in specific cell types in somatic embryos using Lat B treatment accelerated and even synchronized the development of somatic embryos and may be of practical use in biotechnology.

Intranuclear accumulation of plant tubulin in response to low temperature.

Summary.Concurrently with cold-induced disintegration of microtubular structures in the cytoplasm, gradual tubulin accumulation was observed in a progressively growing proportion of interphase nuclei in tobacco BY-2 cells. This intranuclear tubulin disappeared upon rewarming. Simultaneously, new microtubules rapidly emerged from the nuclear periphery and reconstituted new cortical arrays, as was shown by immunofluorescence. A rapid exclusion of tubulin from the nucleus during rewarming was also observed in vivo in cells expressing GFP-tubulin. Nuclei were purified from cells that expressed GFP fused to an endoplasmic-reticulum retention signal (BY-2-mGFP5-ER), and green-fluorescent protein was used as a diagnostic marker to confirm that the nuclear fraction was not contaminated by nuclear-envelope proteins. These purified, GFP-free nuclei contained tubulin when isolated from cold-treated cells, whereas control nuclei were void of tubulin. Furthermore, highly conserved putative nuclear-export sequences were identified in tubulin sequences. These results led us to interpret the accumulation of tubulin in interphasic nuclei, as well as its rapid nuclear export, in the context of ancient intranuclear tubulin function during the cell cycle progression.

A novel, cellulose synthesis inhibitory action of ancymidol impairs plant cell expansion

The co-ordination of cell wall synthesis with plant cell expansion is an important topic of contemporary plant biology research. In studies of cell wall synthesis pathways, cellulose synthesis inhibitors are broadly used. It is demonstrated here that ancymidol, known as a plant growth retardant primarily affecting gibberellin biosynthesis, is also capable of inhibiting cellulose synthesis. Its ability to inhibit cellulose synthesis is not related to its anti-gibberellin action and possesses some unique features never previously observed when conventional cellulose synthesis inhibitors were used. It is suggested that ancymidol targets the cell wall synthesis pathway at a regulatory step where cell wall synthesis and cell expansion are coupled. The elucidation of the ancymidol target in plant cells could potentially contribute to our understanding of cell wall synthesis and cell expansion control.

ARP2 and ARP3 are localized to sites of actin filament nucleation in tobacco BY-2 cells

Summary.Complete depolymerization of actin filaments (AFs) at low temperature (0 °C) is followed by the formation of transient actin structures at 25 °C in tobacco BY-2 cells (Nicotiana tabacum L.). Using antibodies against fission yeast actin-related proteins (ARP2 and ARP3), we show here that transient actin structures (dots, dotted filaments, rods) colocalize with epitopes stained by these antibodies and thus are likely to represent sites of actin filament nucleation (SANs). In contrast to the cold-induced disassembly of AFs, no transient actin structures were detectable during recovery of AFs from latrunculin B-induced depolymerization. However, the staining pattern obtained with ARP antibodies in latrunculin B-treated cells was similar to that in controls and cold-treated cells. This suggests that, in addition to the complete depolymerization of AFs, disruption of other cellular structures is needed for the formation of transient actin structures during the early phase of recovery from cold treatment.

Non-apoptotic programmed cell death with paraptotic-like features in bleomycin-treated plant cells is suppressed by inhibition of ATM/ATR pathways or NtE2F overexpression

In plants, different forms of programmed cell death (PCD) have been identified, but they only partially correspond to those described for animals, which is most probably due to structural differences between animal and plant cells. Here, the results show that in tobacco BY-2 cells, bleomycin (BLM), an inducer of double-strand breaks (DSBs), triggers a novel type of non-apoptotic PCD with paraptotic-like features. Analysis of numerous PCD markers revealed an extensive vacuolization, vacuolar rupture, and chromatin condensation, but no apoptotic DNA fragmentation, fragmentation of the nuclei, or sensitivity to caspase inhibitors. BLM-induced PCD was cell cycle regulated, occurring predominantly upon G(2)/M cell cycle checkpoint activation. In addition, this paraptotic-like PCD was at least partially inhibited by caffeine, a known inhibitor of DNA damage sensor kinases ATM and ATR. Interestingly, overexpression of one NtE2F transcriptional factor, whose homologues play a dual role in animal apoptosis and DNA repair, reduced PCD induction and modulated G(2)/M checkpoint activation in BY-2 cells. These observations provide a solid ground for further investigations into the paraptotic-like PCD in plants, which might represent an ancestral non-apoptotic form of PCD conserved among animals, protists, and plants.

The structure of cortical cytoplasm in cold‐treated tobacco cells:the role of the cytoskeleton and the endomembrane system

The eukaryotic cytoskeleton is a highly dynamic and complex structure supporting many basic metabolic processes. Generally, the organisation of cytoskeleton carries the spatial information important for various cellular compartments and moreover secures the mobility of protein-transporting membrane vesicles. One of the important features of cytoskeleton polymers is their high sensitivity to low (non-freezing) temperatures leading to reversible depolymerisation into subunits. However, cold sensitivity of cytoskeletal polymers differs in various plants and plant tissues depending on many interfering factors (Baluska et al., 1993; Egierszdorff and Kacperska, 2001; Kerr and Carter, 1990). In addition to that, spatial interconnection of cytoskeleton with endomembrane system plays an important role (for review see Lichtscheidl and Baluska, 2000). Coldinduced depolymerisation of both microtubules (MTs) and actin filaments (AFs) provided us with a tool to investigate the role of the cytoskeleton in the spatial organisation of the plant cell cytoplasm. The tobacco BY-2 suspension culture (Nagata et al., 1992) has been used as a model, physiologically homogenous plant cell line. We have focused on the cortical cytoplasm of BY-2 tobacco cells, where structural changes can be easily observed by conventional light and fluorescence microscopy (reviewed in Kumagai and Hasezawa, 2001). Together with this, the organisation of the endoplasmic reticulum (ER) was studied in vivo using BY-2 cells expressing a GFP-fusion protein targeted to the lumen of the ER. Protein analysis of cytoskeleton proteins was carried out to understand some cold-induced cytoskeletal changes on the protein level.

Structure and expression of a hybrid proline-rich protein gene in the solanaceous species,Solanum brevidens, Solanum tuberosum, andLycopersicum esculentum

Summary The full-length cDNA of a previously identified Solanum brevidens gene was isolated and characterised. DNA sequence analysis revealed an open reading frame that encodes a hybrid proline-rich cell wall protein of 407 amino acids. The putative protein was designated SbrPRP. The SbrPRP harbours three parts, an N-terminal signal peptide followed by a repetitive proline-rich domain and a cysteine-rich C-terminus resembling non-specific lipid-transfer proteins. The repetitive proline-rich domain contains two repeated motifs, PPHVKPPSTPK and PTPPIVSPP extended with TPKYP and TPKPPS motifs, respectively, at their N- or C-terminal. The SbrPRP gene of the non-tuberising Solanum species, Solanum brevidens , possesses highly homologous counterparts in the tuberising species, Solanum tuberosum ( StPRP ) and in the related species, Lycopersicum esculentum ( TFM7 ). All three genes are present in single- or low copy number in the corresponding genome. Organ-specific expression of the genes, however, is different in the three solanaceous species.

Plant Cell Strains in Fundamental Research and Applications

This chapter introduces plant cell strains and describes their main characteristics which render them unique experimental models to study plant morphogenesis. Although composed only of few cells, these systems are characterised by strict natural polarity of cell growth and division and high spontaneous friability of the daughter cell files. Both features are most clearly exhibited in two tobacco cell lines, Virginia Bright Italia (VBI-0) and Bright Yellow (BY-2). Owing to their ability to grow in vitro resembling animal single cell layer systems, they offer the possibility to accompany molecular or biochemical analyses of the material by a precise but nondestructive in vivo cytological observation of the cell population. Here we summarise the use of these two lines in various plant phenotyping studies including investigations of morphoregulatory signals like phytohormones or stress factors as well as organisation of cytoskeleton, endomembranes and cell wall. Although so far not frequently utilised in practical applications, some transgenic BY-2 strains show high potential for molecular farming. Last but not the least, the mechanisms responsible for the spontaneous or targeted selection of similar plant cell strains from genetically more attractive plant species are discussed.

Plant Cell Lines in Cell Morphogenesis Research

Plant organs and tissues consist of many various cell types, often in different phases of their development. Such complex structures do not allow direct studies on behavior of individual cells. In contrast, populations of in vitro-cultured plant cells represent valuable tool for studying processes on a single-cell level, including cell morphogenesis. Here we describe characteristics of well-established model tobacco and Arabidopsis cell lines and provide detailed protocol on their cultivation, characterization, and genetic transformation.