In Vivo Dynamics and Differential Microtubule-Binding Activities of MAP65 Proteins

Abstract

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...

Two Microtubule-associated Proteins of Arabidopsis MAP65s Promote Antiparallel Microtubule Bundling

“…Typically, during interphase CMTs were decorated with GFP-AtMAP65-5 (Van Damme et al, 2004a). We found that during prophase, GFPAtMAP65-5 strongly labeled the preprophase band, a hallmark for the division zone, and the perinuclear MTs before the initiation of the bipolar spindle ( Figure 7A, Video S1).…”

“…AtMAP65-6 colocalizes with mitochondria (Mao et al, 2005b). Finally, AtMAP65-5 associates with a subset of transversely organized CMT bundles in interphase as well as the phragmoplast during cytokinesis (Van Damme et al, 2004a). On the basis of the their diverse localization patterns, we postulate that AtMAP65s exhibit differential molecular and functional properties which still remain to be identified.…”

“…AtMAP65-3 is associated with the mitotic MT array during both early and late mitosis in all Arabidopsis organs (Caillaud et al, 2008). AtMAP65-4 associates with the prophase spindle MTs and labels the spindle MTs until anaphase (Van Damme et al, 2004a). AtMAP65-6 colocalizes with mitochondria (Mao et al, 2005b).…”

“…AtMAP65-1-GFP, AtMAP65-5-GFP, and red fluorescent protein (RFP)-TUA6 constructs are already described (Van Damme et al, 2004a). AtMAP65-GFP constructs were supertransformed into a BY-2 suspension culture stably expressing RFP-TUA6 as described (Geelen and Inzé, 2001).…”

“…Overall sequence identity is poorly conserved, suggesting that AtMAP65 proteins have adopted separate properties. AtMAP65-1 is so far the more studied and was shown to colocalize with bundled CMTs and the spindle midzone in anaphase (Van Damme et al, 2004a;Mao et al, 2005a;Smertenko et al, 2006). In vitro, AtMAP65-1 promotes MT bundling and appears as filamentous cross-bridges regularly spaced along the MT wall (Smertenko et al, 2004;Mao et al, 2005b).…”