Three-dimensional fluorescence microscopy.
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Microscopy, Fluorescence↗
Biomedical subjects
D J Rawlins
Publications and source records attributed to D J Rawlins.
Nucleus-associated microtubules help determine the division plane of plant epidermal cells: avoidance of four-way junctions and the role of cell geometry.
To investigate the spatial relationship between the nucleus and the cortical division site, epidermal cells were selected in which the separation between these two areas is large. Avoiding enzyme treatment and air drying, Datura stramonium cells were labeled with antitubulin antibodies and the three-dimensional aspect of the cytoskeletons was reconstructed using computer-aided optical sectioning. In vacuolated cells preparing for division, the nucleus migrates into the center of the cell, suspended by transvacuolar strands. These strands are now shown to contain continuous bundles of microtubules which bridge the nucleus to the cortex. These nucleus-radiating microtubules adopt different configurations in cells of different shape. In elongated cells with more or less parallel side walls, oblique strands radiating from the nucleus to the long side walls are presumably unstable, for they are progressively realigned into a transverse disc (the phragmosome) as broad, cortical, preprophase bands (PPBs) become tighter. The phragmosome and the PPB are both known predictors of the division plane and our observations indicate that they align simultaneously in elongated epidermal cells. These observations suggest another hypothesis: that the PPB may contain microtubules polymerized from the nuclear surface. In elongated cells, the majority of the radiating microtubules, therefore, come to anchor the nucleus in the transverse plane, consistent with the observed tendency of such cells to divide perpendicular to the long axis. In nonrectangular isodiametric epidermal cells, which approximate regular hexagons in section, the radial microtubular strands emanating from the nucleus tend to remain associated with the middle of each subtending cell wall. The strands are not reorganized into a single dominant transverse bar, but remain as a starlike array until mitosis. PPBs in these cells are not as tight; they may only be a sparse accumulation of microtubules, even forming along non-diametrical radii. This arrangement is consistent with the irregular division patterns observed in epidermal mosaics of isodiametric D. stramonium cells. The various conformations of the radial strands can be modeled by springs held in two-dimensional hexagonal frames, and by soap bubbles in three-dimensional hexagonal frames, suggesting that the division plane may, by analogy, be selected by minimal path criteria. Such behavior offers a cytoplasmic explanation of long-standing empirically derived "rules" which state that the new cell wall tends to meet the maternal wall at right angles. The radial premitotic strands and their analogues avoid taking the longer path to the vertex of an angle where a cross wall is already present between neighboring cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Cell Division↗
Localization of ribosomal and telomeric DNA sequences in intact plant nuclei by in-situ hybridization and three-dimensional optical microscopy.
We have combined the use of three-dimensional (3-D) fluorescence microscopy and computer image processing of images with in-situ hybridization to analyse the 3-D organization of interphase nuclei in plants. In sections of root tips of Pisum sativum, using cDNA probes, we have shown that telomeres are arranged around the nuclear periphery and that the ribosomal genes in this species appear to exist in discrete, 3-D domains.
Cell Nucleus↗
Three-dimensional localisation of DNA in the nucleolus of Spirogyra by correlated optical tomography and serial ultra-thin sectioning.
Spirogyra nucleoli were shown by three-dimensional optical microscopy of DAPI fluorescence to contain DNA with a pattern and distribution matching those of the fibrillar centres. This was confirmed using different species with nucleoli showing different sizes of fibrillar centre. Much lower levels of fluorescence were seen corresponding to the dense fibrillar component. Nearly all the DAPI fluorescence arises from the fibrillar centres or from regions very close to their surface, indicating that this is the site of nucleolar transcription.
Cell Nucleolus↗
Three-dimensional organization of chromosomes of Crepis capillaris by optical tomography.
The three-dimensional (3D) organization of chromosomes of Crepis capillaris (2n = 6) has been investigated. Root tips were fixed, macerated with enzymes and gently separated without squashing. The cells were then stained with DAPI and optically sectioned under computer control. Sections were stored as video images and processed to remove noise and out-of-focus information. Computer modelling was then used to trace the paths of each chromosome and to display the paths as a 3D wire diagram. In all, 88 sets of anaphase chromosomes were modelled from 47 optically sectioned cells. The models and the coordinates of the chromosomes were then analysed to detect non-random arrangements or preferential associations of particular pairs of chromosomes. The methods used have significant advantages over electron microscope tomography for the analysis of 3D chromosome arrangement; in particular, the large number of samples allowed more thorough statistical tests to be performed on the data obtained. No evidence was found for either non-random arrangements or homologous association and, moreover, the distances between the two larger pairs of homologues were larger than for other pairs of chromosomes. These results conflict with previous results for this and other plant species where the material was squashed before measurements were taken. We found no evidence of haploid genome separation.
Chromosomes↗
An actin network is present in the cytoplasm throughout the cell cycle of carrot cells and associates with the dividing nucleus.
We have studied the F-actin network in cycling suspension culture cells of carrot (Daucus carota L.) using rhodaminyl lysine phallotoxin (RLP). In addition to conventional fixation with formaldehyde, we have used two different nonfixation methods before adding RLP: extracting cells in a stabilizing buffer; inducing transient pores in the plasma membrane with pulses of direct current (electroporation). These alternative methods for introducing RLP revealed additional features of the actin network not seen in aldehyde-fixed cells. The three-dimensional organization of this network in nonflattened cells was demonstrated by projecting stereopairs derived from through-focal series of computer-enhanced images. F-actin is present in interphase cells in four interconnected configurations: a meshwork surrounding the nucleus; thick cables in transvacuolar strands and deep in the cytoplasm; a finer network of bundles within the cortical cytoplasm; even finer filaments that run in ordered transverse array around the cell periphery. The actin network is organized differently during division but it does not disappear as do the cortical microtubules. RLP stains a central filamentous cortical band as the chromatin begins to condense (preprophase); it stains the mitotic spindle (as recently shown by Seagull et al. [Seagull, R. W., M. Falconer, and C. A. Weerdenburg, 1987, J. Cell Biol., 104:995-1004] for aldehyde fixed suspension cells) and the cytokinetic apparatus (as shown by Clayton, L., and C. W. Lloyd, 1985, Exp. Cell Res., 156:231-238). However, it is now shown that an additional network of F-actin persists in the cytoplasm throughout division associating in turn with the preprophase band, the mitotic spindle, and the cytokinetic phragmoplast.
Actin Cytoskeleton↗