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Centrin - higher plants - MTOCs - microtubules nucleation In most eukaryotic cells, the Ca(2+)-binding protein centrin is associated with structured microtubule-organizing centers (MTOCs) such as centrosomes. In these cells, centrin either forms centrosome-associated contractile fibers, or is involved in centrosome biogenesis. Our aim was to investigate the functions of centrin in higher plant cells which do not contain centrosome-like MTOCs. We have cloned two tobacco BY-2 centrin cDNAs and we show that higher plant centrins define a phylogenetic group of proteins distinct from centrosome-associated centrins. In addition, tobacco centrins were found primarily associated with microsomes and did not colocalize with gamma-tubulin, a known MTOC marker. While the overall level of centrin did not vary during the cell cycle, centrin was prominently detected at the cell plate during telophase. Our results suggest that in tobacco, the major portion of centrin is not MTOC-associated and could be involved in the formation of the cell plate during cytokinesis.

Compelling evidence supports the idea that actin filaments play an active role in the cytokinetic process of higher plant cells. However, the mechanisms that control the growth of the cell plate and its stabilization remain so far unknown. We show that a novel population of short actin filaments continuously assembles in the phragmoplast at the growing cell plate. Microinjection of rhodamine-phalloidin during these final stages of telophase revealed the dynamic assembly and organization of these actin filaments during vesicle fusion. Comparable data were obtained in endosperm syncytia during the development of the cell plate between non sister nuclei, i.e. independently of the formation of the mitotic phragmoplast. Concomitantly, plant polypeptides sharing epitopes with human vinculin are revealed within the forming cell plate, suggesting their recruitment during cytokinesis-associated actin assembly. These vinculin-like antigens may participate in membrane/F-actin anchorage protein complexes. Our data, in addition to the identification of plant integrin homologues reported by several authors, suggest the existence of a cell wall/extracellular matrix/plasma membrane/actin cytoskeleton continuum. Such an architecture may control cell-cell interactions during cell plate formation and may contribute to the establishment of polarity in higher plants.

In this study, we investigated the effect of plant microtubule-associated proteins (MAPs) on microtubule nucleation and growth in vitro. Since it has recently been demonstrated that plant nuclear surface acts as a microtubule-organizing center (MTOC), we tested the effects of plant MAPs using a nucleus-mediated microtubule nucleation assay. Nuclei were isolated from interphase tobacco BY-2 cells, and MAPs were isolated from tobacco BY-2 cells at different stages of the cell cycle. The effects of tobacco MAPs on microtubule nucleation at mammalian centrosomes were also analyzed. Under our experimental conditions, both interphase and mitotic tobacco MAPs promoted microtubule assembly around tobacco nuclei and at mammalian centrosomes below the critical tubulin concentration for spontaneous assembly. Interphase tobacco MAPs increase the mean length of nucleated microtubules in proportion to its molar ratio to tubulin. In contrast, mitotic tobacco MAPs do not induce nucleus- and centrosome-mediated nucleation of microtubules in a dose-dependent manner. Both MAP-fractions possessed microtubule bundling activity. The implications of these plant MAP properties on microtubule nucleation in living cells are discussed.