SummaryTo identify molecular players implicated in cytokinesis and division plane determination, the Arabidopsis thaliana genome was explored for potential cytokinesis genes. More than 100 open reading frames were selected based on similarity to yeast and animal cytokinesis genes, cytoskeleton and polarity genes, and Nicotiana tabacum genes showing cell cycle-controlled expression. The subcellular localization of these proteins was determined by means of GFP tagging in tobacco Bright Yellow-2 cells and Arabidopsis plants. Detailed confocal microscopy identified 15 proteins targeted to distinct regions of the phragmoplast and the cell plate. EB1-and MAP65-like proteins were associated with the plus-end, the minus-end, or along the entire length of microtubules. The actin-binding protein myosin, the kinase Aurora, and a novel cell cycle protein designated T22, accumulated preferentially at the midline. EB1 and Aurora, in addition to other regulatory proteins (homologs of Mob1, Sid1, and Sid2), were targeted to the nucleus, suggesting that this organelle operates as a coordinating hub for cytokinesis.

Plant cells produce different microtubule arrays that are essential for cell division and morphogenesis without equivalent in other eukaryotes. Microtubule-associated proteins influence the behavior of microtubules that is presumed to culminate into transitions from one array to another. We analyzed the microtubule-binding properties of three Arabidopsis (Arabidopsis thaliana) members, AtMAP65-1, AtMAP65-4, and AtMAP65-5, in live cells using laser scanning confocal microscopy. Depending on the overall organization of the cortical array, AtMAP65-1-GFP (green fluorescent protein) and AtMAP65-5-GFP associated with a subset of microtubules. In cells containing both coaligned and oblique microtubules, AtMAP65-1-GFP and AtMAP65-5-GFP tended to be associated with the coaligned microtubules. Cortical microtubules labeled with AtMAP65-1-GFP and AtMAP65-5-GFP appeared as thick bundles and showed more resistance to microtubule-destabilizing drugs. The polymerization rates of AtMAP65-1-GFP and AtMAP65-5-GFP microtubules were similar to those of tubulin-GFP marked microtubules but were different from AtEB1a-GFP, a microtubule plus-end-binding EB1-like protein that stimulated polymerization. By contrast, depolymerization rates of AtMAP65-1-GFP-and AtMAP65-5-GFP-labeled microtubules were reduced. AtMAP65-1-GFP associated with polymerizing microtubules within a bundle, and with fixed microtubule termini, suggesting that AtMAP65-1's function is to bundle and stabilize adjacent microtubules of the cortex. Polymerization within a bundle took place in either direction so that bundling occurred between parallel or antiparallel aligned microtubules. AtMAP65-4-GFP did not label cortical microtubules or the preprophase band, despite continuous expression driven by the 35S promoter, and its subcellular localization was restricted to microtubules that rearranged to form a spindle and the polar sides of the spindle proper. The expression of AtMAP65-4 peaked at mitosis, in agreement with a function related to spindle formation, whereas AtMAP65-1 and AtMAP65-5 were expressed throughout the cell cycle.Microtubules are polar filamentous structures with a highly dynamic plus end and a more stable minus end. The plus end shows alternating phases of growth (polymerization) and rapid shortening (depolymerization), a phenomenon that is also known as dynamic instability. The dynamic instability of microtubules depends on two couples of mutually excluding parameters: the growth and shrinkage rate, and the frequencies at which a microtubule undergoes transitions between polymerization (growth phase) and depolymerization (shrinkage). The transition from a growing phase to shrinkage is termed catastrophy and from shrinkage phase to a growing phase termed rescue. The minus end is associated with microtubule organizing centers that nucleate microtubules. Microtubules are arranged into different arrays implicated in cell division and differentiation and hence are subject to various reorganizations. Plants have three unique microtubule arrays: the cortical...

SummaryGiant cells induced by root knot nematodes and syncytia caused by cyst nematodes are large multinucleated feeding cells containing a dense cytoplasm generated during a complex host±parasite association in plant roots. To ®nd out whether cytoskeleton changes occurred during feeding cell development, transcriptional activity of actin (ACT) and tubulin genes and organization of the ACT ®laments and of the microtubules (MTs) were analyzed in situ. The importance of changes in the cytoskeleton architecture for the proper initiation and development of galls and syncytia was demonstrated by perturbing the cytoskeleton with chemical inhibitors. The expression levels of cytoskeletal components, such as tubulins and ACTs, are proposed to be upregulated to allow the assembly of a new cytoskeleton in expanding feeding cells. However, MTs and ACT ®laments failed to properly organize and appeared partially depolymerized throughout feeding site development. Both the actin and tubulin cytoskeletons were strongly disrupted in syncytia and mitotic ®gures were never observed. In contrast, in giant cells, an ACT and cortical MT cytokeleton, although disturbed, was still visible. In addition, a functional mitotic apparatus was present that contained multiple large spindles and arrested phragmoplasts, but no pre-prophase bands. Chemical stabilization of the microtubular cytoskeleton with taxol blocked feeding site development. On the other hand, when the ACT or MT cytoskeleton of feeding cells was depolymerized by cytochalasin D or oryzalin, nematodes could complete their life cycle. Our data suggest that the cytoskeleton rearrangements and depolymerization induced by parasitic nematodes may be essential for a successful feeding process.