On the distribution of microtubule-associated intermediate filament antigens in plant suspension cells

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Abstract
Intermediate filament antigens are known to coalign, patchily, with cortical microtubules in plant cells, but nothing else is established about this relationship or the form the antigens take. This was studied further using cell suspensions, instead of root tip cells, since their cortex is accessible to antibodies without the rigours of cell separation. Both carrot and sweetcorn suspension cells were labelled with three antibodies known to recognize animal intermediate filaments. The mitotic and cytokinetic apparatus could be stained with these antibodies, which, in interphase cells, also labelled cortical microtubule-like arrays. One antibody (AFB), raised against cytoplasmic bundles of 7nm fibrils from carrot cells, immunostained the bundles but only under conditions of fixation that did not allow the finer, microtubule-associated staining pattern to be seen. By exploring various preparatory conditions it was concluded that these two forms of antigen co-exist: they are not experimentally interconvertible but require different conditions for exposure to different antibodies. Double immunofluorescence established that the intermediate filament antigens do not parallel the actin network, nor did cytochalasin D affect their distribution. Taxol, however, bundled the intermediate filament antigens, whereas they are dispersed when microtubules are depolymerized, rather than collapsing in perinuclear whorls. Under conditions permitting the microtubule associated antigens to be stained by immunofluorescence, carrot protoplasts were cleaved on grids, exposing the cortical microtubules. Immunogold labelling then showed that the antibody raised against fibrillar bundles recognizes patches of electron-dense material, along and between the microtubules, rather than individual filaments. To confirm that the plant antigens are capable of forming filaments, a high salt, detergent-insoluble fraction was prepared from the maize line. By dialysing from urea, intermediate-sized filaments could be reconstituted and they immunoblotted with the broadly cross-reactive antibody to intermediate filament antigens (anti-IFA). These studies underline the problems of visualizing in plants, conformations of intermediate filament antigens that are not directly comparable to the extensively studied animal models, and that appear to be sensitive to the way in which cells are manipulated.