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The assembly of the higher plant cytoskeleton poses several fundamental questions. Since different microtubule arrays are successively assembled during the cell cycle in the absence of centrosomes, we can ask how these arrays are assembled and spatially organized. Two hypotheses are under debate. Either multiple nucleation sites are responsible for the assembly and organization of microtubule arrays or microtubule nucleation takes place at one site, the nuclear surface. In the latter case, microtubule nucleation and organization would be two distinct but coregulated processes. During recent years, novel approaches have provided entirely new insights to understand the assembly and dynamics of the plant cytoskeleton. In the present review, we summarize advances made in microscopy and in molecular biology which lead to novel hypotheses and open up new fields of investigation. From the results obtained, it is clear that the higher plant cell is a powerful model system to investigate cytoskeletal organization in acentrosomal eukaryotic cells., (Copyright 2000 Wiley-Liss, Inc.)

Plants can adapt their shape to environmental stimuli. This response is mediated by the reorganization of cortical microtubules, a unique element of the cytoskeleton. However, the molecular base of this response has remained obscure so far. In an attempt to solve this problem, signal-dependent changes in the pattern of microtubule-binding proteins were analysed during coleoptile elongation in maize, that is, under the control of the plant photoreceptor phytochrome. Two putative MAPs of 100 kDa (P100) and 50 kDa apparent molecular weights were identified in cytosolic extracts from non-elongating and elongating cells. Both proteins co-assembled with endogenous tubulin, bound to neurotubules and were immunologically related to the neural MAP tau: the P100 protein, depending on the physiological situation, was manifest as a double band and was always found to be heat-stable. In contrast, the 50 kDa MAP was heat-stable only for particular tissues and physiological treatments. The P100 protein was present in all tissues, however in a reduced amount in elongating coleoptiles. The 50 kDa MAP was expressed exclusively upon induction of phytochrome-dependent cell elongation. As shown by immunofluorescence double-staining, an epitope shared by both proteins colocalized with cortical microtubules in situ, but exclusively in elongating cells. In non-elongating cells, only the nuclei were stained. Partially purified nuclei from elongating cells were enriched in P100, whereas the 50 kDa MAP became enriched in a partially purified plasma membrane fraction.

Recent developments of immunotherapeutic approaches have shown that artificial ordering of tumor cell membranes with cholesterol hemisuccinate (CHS) or 25-hydroxycholesterol (25-OH) may significantly enhance the immunogenicity of human renal adenocarcinoma cells. To gain further insight into the molecular mechanism of these sterols, we investigated cytoskeletal modification, which is related to the cell membrane. After treatment of human renal carcinoma cells with these cholesterol (at 10(-6) and 10(-7) M) for 5 days, we observed a disorganization of the submembrane end of the cytoplasmic actin stress fibers by cytofluorescence. The microtubule network was not affected. Thus, in the present study, we found that changes in membrane physicochemical properties impaired the anchorage of actin microfilaments in the plasma membrane of human renal cancer cells. Under the same experimental conditions, such modifications were not observed in normal cells (human fibroblasts) or in human hepatoma cells. We suggest that incubation of cancer cells with these sterols induced a redistribution of the cholesterol-rich membrane microdomains which are linked to the cytoskeleton through submembrane proteins.

We have identified an F-actin cytoskeletal network that remains throughout interphase, mitosis, and cytokinesis of higher plant endosperm cells. Fluorescent labeling was obtained using actin monoclonal antibodies and/or rhodamine-phalloidin. Video-enhanced microscopy and ultrastructural observations of immunogold-labeled preparations illustrated microfilament-microtubule co-distribution and interactions. Actin was also identified in cell crude extract with Western blotting. During interphase, microfilament and microtubule arrays formed two distinct networks that intermingled. At the onset of mitosis, when microtubules rearranged into the mitotic spindle, microfilaments were redistributed to the cell cortex, while few microfilaments remained in the spindle. During mitosis, the cortical actin network remained as an elastic cage around the mitotic apparatus and was stretched parallel to the spindle axis during poleward movement of chromosomes. This suggested the presence of dynamic cross-links that rearrange when they are submitted to slow and regular mitotic forces. At the poles, the regular network is maintained. After midanaphase, new, short microfilaments invaded the equator when interzonal vesicles were transported along the phragmoplast microtubules. Colchicine did not affect actin distribution, and cytochalasin B or D did not inhibit chromosome transport. Our data on endosperm cells suggested that plant cytoplasmic actin has an important role in the cell cortex integrity and in the structural dynamics of the poorly understood cytoplasm-mitotic spindle interface. F-actin may contribute to the regulatory mechanisms of microtubule-dependent or guided transport of vesicles during mitosis and cytokinesis in higher plant cells.