In plants, it is unclear how dispersed cortical microtubules are nucleated, polarized and organized in the absence of centrosomes. In Arabidopsis thaliana cells, expression of a fusion between the microtubule-end-binding protein AtEB1a and green fluorescent protein (GFP) results in labelling of spindle poles, where minus ends gather. During interphase, AtEB1a-GFP labels the microtubule plus end as a comet, but also marks the minus end as a site from which microtubules can grow and shrink. These minus-end nucleation sites are mobile, explaining how the cortical array can redistribute during the cell cycle and supporting the idea of a flexible centrosome in plants.

The 65-kD microtubule-associated protein (MAP65) family is a family of plant microtubule-bundling proteins. Functional analysis is complicated by the heterogeneity within this family: there are nine MAP65 genes in Arabidopsis thaliana, AtMAP65-1 to AtMAP65-9. To begin the functional dissection of the Arabidopsis MAP65 proteins, we have concentrated on a single isoform, AtMAP65-1, and examined its effect on the dynamics of mammalian microtubules. We show that recombinant AtMAP65-1 does not promote polymerization and does not stabilize microtubules against cold-induced microtubule depolymerization. However, we show that it does induce microtubule bundling in vitro and that this protein forms 25-nm cross-bridges between microtubules. We further demonstrate that the microtubule binding region resides in the C-terminal half of the protein and that Ala409 and Ala420 are essential for the interaction with microtubules. Ala420 is a conserved amino acid in the AtMAP65 family and is mutated to Val in the cytokinesis-defective mutant pleiade-4 of the AtMAP65-3/PLEIADE gene. We show that AtMAP65-1 can form dimers and that a region in the N terminus is responsible for this activity. Neither the microtubule binding region nor the dimerization region alone could induce microtubule bundling, strongly suggesting that dimerization is necessary to produce the microtubule cross-bridges. In vivo, AtMAP65-1 is ubiquitously expressed both during the cell cycle and in all plant organs and tissues with the exception of anthers and petals. Moreover, using an antiserum raised to AtMAP65-1, we show that AtMAP65-1 binds microtubules at specific stages of the cell cycle.

Xylem tracheary elements (TEs) form hollow, sap-conducting tubes kept open by thickened ribs of secondary cell wall that provide the major structural element in wood. These ribs are enriched with cellulose and lignin, molecules that utilize more atmospheric CO(2) than any other biopolymer on Earth. The thickenings form characteristic patterns (e.g., spiral and pitted) that depend upon the bundling of underlying microtubules [1, 2]. To identify microtubule-associated proteins (MAPs) involved in patterning microtubules, we optimized an in vitro system for triggering single Arabidopsis cells to differentiate synchronously into TEs. From more than 200 microtubule-implicated proteins, AtMAP70-5 was the only MAP upregulated upon, and specific to, TE differentiation. It lines the borders of each microtubule bundle and forms C-shaped "spacers" between adjacent bundles. Manipulating levels of AtMAP70-5 and its binding partner AtMAP70-1 by overexpression or RNA interference (RNAi) silencing shifted the balance between the characteristic patterns. RNAi silencing produced stunted plants with disorganized vascular bundles. In culture, RNAi knockdown caused ribs of secondary cell wall, surrounded by microtubules, to invaginate and fall into the cytoplasm. These results suggest that AtMAP70-5 and AtMAP70-1 are essential for defining where secondary cell wall polymers are applied at the cell cortex in wood-forming cells.

Abstract. We have studied the F-actin network in cycling suspension culture cells of carrot (Daucus carom L.) using rhodaminyl lysine phallotoxin (RLP).In addition to conventional fixation with formaldehyde, we have used two different nonfixation methods before adding RLP: extracting cells in a stabilizing buffer; inducing transient pores in the plasma membrane with pulses of direct current (electroporation). These alternative methods for introducing RLP revealed additional features of the actin network not seen in aldehyde-fixed cells. The three-dimensional organization of this network in nonflattened cells was demonstrated by projecting stereopairs derived from through-focal series of computer-enhanced images.