SummaryThis article describes a practical technique for permeabilization of higher plant cell walls, which is usually one of the first steps required for immunolocalization of cellular components (and other cytological methods) in plant cell studies. Our strategy involves shattering the walls of cells while the tissues are frozen in liquid nitrogen. It replaces the use of wall degrading enzymes or the need to employ laborious sectioning or other mechanical means for providing access of probes to cells. Freeze-shattering retains the integrity of whole tissues and cells surprisingly well and thus is especially useful when used in conjunction with confocal laser scanning microscopy for recording the threedimensional arrangement of cytoskeletal elements in relation to cell shape. In this article, we demonstrate the effectiveness of this technique for anti-tubulin and antiactin immunofluorescence and for rhodamine phalloidin labelling of the cytoskeleton in various higher plant tissues including onion root tip and bulb scale epidermis, Tradescantia stamen hairs and Arabidopsis leaf epidermis and mesophyll cells.

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Ehrlich ascites tumor (EAT) cells propagated in mass suspension culture were used as a starting material for the simultaneous isolation and purification of large quantities of the intermediate filament protein vimentin and the nuclear lamins A/C and B. Triton cytoskeletons, obtained by repeated washing of cells with a low ionic strength buffer containing Triton X-100 and 4 mM Mg2+, were extracted with 6 M urea at low salt concentration and in the presence of EDTA. Separation of solubilized proteins from unfolded chromatin (DNA) was accomplished by recondensation of the chromatin (DNA) in the presence of Mg2+ before centrifugation. To achieve separation of vimentin from nuclear lamins, the urea extract was subjected to DEAE-Sepharose CL-6B chromatography. Single-stranded DNA-cellulose chromatography was employed for the final purification of vimentin and for the separation of lamin B from lamins A/C. Further purification of lamin B was carried out by CM-Sepharose CL-6B chromatography and of lamins A/C by chromatography on hydroxylapatite. All chromatographies were performed in the presence of 6 M urea. 500 g of pelleted EAT cells yielded approximately 700 mg of vimentin, 225 mg of lamins A/C and 21 mg of lamin B. 2D-polyacrylamide gel electrophoresis revealed great microheterogeneity of lamins A/C, which to a high extent was due to phosphorylation, whereas lamin B was much less heterogeneous. In the absence of urea and at low salt concentration, lamins A/C required pH 5 to stay in solution whereas lamin B required pH 7.5. Increasing the salt concentration to 150 or 250 mM NaCl resulted in the formation of paracrystals from a urea-free mixture of lamins A/C and B. Although the lamins could not be assembled into intermediate filaments under a variety of ionic conditions, the preparations obtained will be useful for further biochemical characterization of these nuclear proteins.

Employing indirect immunofluorescence and conventional electron microscopy, gross nuclear aberrations were observed in cultured interphase fibroblasts derived from a patient suffering from Fanconi's anemia (FA). Such aberrations were predominantly expressed in cells at high passages between 28 and 34. The structure of the nuclei appeared compound in nature, often consisting of two to three nuclear fragments connected to each other by thin nuclear bridges containing chromatin and nuclear lamin material. In other cases, the nuclei appeared lobed or budded but the cells did not contain distinct nuclear fragments. Chromatin was conspicuously absent from some nuclear lobes, revealing empty, cage-like structures comprising nuclear lamin material. Micronuclei were often abundant in the perinuclear cytoplasm but in some instances they appeared to be composed of chromatin lacking a delineating nuclear lamin matrix. Residual cytoskeletons examined by whole-mount electron microscopy revealed a network of intermediate filaments (IFs) within FA fibroblasts forming a bridge between the plasma membrane and the nucleus or its major fragments. In addition, there were thinner, 3–4 nm filaments connecting individual IFs with the surface of the nucleus. Micronuclei that were not connected to the main nuclear body, but which were delineated by a distinct lamina and possessed nuclear pores, did not appear to be anchored to the IF network. Multinuclearity, nuclear fragmentation, irregular chromatin distribution and inter-nuclear chromatin/lamin bridges might result from a failure in the redistribution of chromatin to sister nuclei, incomplete cytokinesis and proliferation of nuclear envelope material. These phenomena point to precocious aging of FA fibroblasts and may occur as a consequence of spontaneous damage to the sister chromatids or through the action of DNA-toxic agents.