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

Complete depolymerization of actin filaments (AFs) at low temperature (0 degrees C) is followed by the formation of transient actin structures at 25 degrees 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.

Intranuclear accumulation of plant tubulin in response to low temperature.

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.

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.

Post-translational modifications and multiple tubulin isoforms in Nicotiana tabacum L. cells.

Distribution of post-translationally modified tubulins in cells of Nicotiana tabacum L. was analysed using a panel of specific antibodies. Polyglutamylated, tyrosinated, nontyrosinated, acetylated and delta 2-tubulin variants were detected on alpha-tubulin subunits; polyglutamylation was also found on beta-tubulin subunits. Modified tubulins were detected by immunofluorescence microscopy in interphase microtubules, preprophase bands, mitotic spindles as well as in phragmoplasts. They were, however, located differently in the various microtubule structures. The antibodies against tyrosinated, acetylated and polyglutamylated tubulins gave uniform staining along all microtubules, while antibodies against nontyrosinated and delta 2-tubulin provided dot-like staining of interphase microtubules. Additionally, immunoreactivity of antibodies against acetylated and delta 2-tubulins was strong in the pole regions of mitotic spindles. High-resolution isoelectric focusing revealed 22 tubulin charge variants in N. tabacum suspension cells. Immunoblotting with antibodies TU-01 and TU-06 against conserved antigenic determinants of alpha- and beta-tubulin molecules, respectively, revealed that 11 isoforms belonged to the alpha-subunit and 11 isoforms to the beta-subunit. Whereas antibodies against polyglutamylated, tyrosinated and acetylated tubulins reacted with several alpha-tubulin isoforms, antibodies against nontyrosinated and delta 2-tubulin reacted with only one. The combined data demonstrate that plant tubulin is extensively post-translationally modified and that these modifications participate in the generation of plant tubulin polymorphism.

Partial auxin deprivation affects endogenous cytokinins in an auxin-dependent, cytokinin-independent tobacco cell strain.

The dynamics of individual endogenous cytokinins within the growth cycle (subculture interval) of an auxin-dependent and cytokinin-independent cell suspension culture ofNicotiana tabacum L. (strain VBI-0) were determined using high performance liquid chromatography and radioimmunoassay. In cells grown at an optimum auxin concentration the transient maxima of N(6)-(Δ(2)-isopentenyl)adenine and N(6)-(Δ(2)-isopentenyl)-adenosine correlated with the onset of cell division. Cultivation of the cells in a partially auxin-deprived medium resulted in ca. tenfold increase of all endogenous cytokinins. A very distinct maximum of N(6)-(Δ(2)-sopentenyl) adenine appeared at the beginning of subculture. This indicates that a lack of auxin induced an accumulation of cytokinins predominantly in the form of the free bases, which are physiologically more active than the corresponding ribosides.