Affinity Purification and Characterization of Functional Tubulin from Cell Suspension Cultures of Arabidopsis and Tobacco

Abstract

Microtubules assemble into several distinct arrays that play important roles in cell division and cell morphogenesis. To decipher the mechanisms that regulate the dynamics and organization of this versatile cytoskeletal component, it is essential to establish in vitro assays that use functional tubulin. Although plant tubulin has been purified previously from protoplasts by reversible taxol-induced polymerization, a simple and efficient purification method has yet to be developed. Here, we used a Tumor Overexpressed Gene (TOG) column, in which the tubulin-binding domains of a yeast (Saccharomyces cerevisiae) TOG homolog are immobilized on resin, to isolate functional plant tubulin. We found that several hundred micrograms of pure tubulin can readily be purified from cell suspension cultures of tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana). The tubulin purified by the TOG column showed high assembly competence, partly because of low levels of polymerization-inhibitory phosphorylation of a-tubulin. Compared with porcine brain tubulin, Arabidopsis tubulin is highly dynamic in vitro at both the plus and minus ends, exhibiting faster shrinkage rates and more frequent catastrophe events, and exhibits frequent spontaneous nucleation. Furthermore, our study shows that an internal histidine tag in a-tubulin can be used to prepare particular isotypes and specifically engineered versions of a-tubulin. In contrast to previous studies of plant tubulin, our mass spectrometry and immunoblot analyses failed to detect posttranslational modification of the isolated Arabidopsis tubulin or detected only low levels of posttranslational modification. This novel technology can be used to prepare assembly-competent, highly dynamic pure tubulin from plant cell cultures.Microtubules (MTs) are important cytoskeletal polymers that are conserved in eukaryotic cells and are assembled from a-and b-tubulin heterodimers (Desai and Mitchison, 1997). In plants, MTs have important functions in essential cellular processes, such as cell division, and in cell morphogenesis. MTs in plant cells adopt several distinct higher order arrays and are remodeled in response to the cell cycle, developmental programs, and environmental cues (Hashimoto, 2015). Genetic, molecular, and cell biological approaches have been used to identify cellular factors that regulate the organization and dynamics of plant MTs. Considerable effort has been devoted to simulating the organization of cortical MT arrays by computational modeling.Cell-free in vitro studies are essential for the biochemical characterization of various MT regulators and for elucidating the mechanistic principles underlying the versatility of this dynamic polymer in cellular functions. The purification of sufficient amounts of assembly-competent tubulin is a prerequisite for these in vitro studies. Tubulin is traditionally purified from mammalian brains, since these tissues contain sufficiently high concentrations of tubulin to allow MT assembly in crude cell extracts. Polymerized MT...

Plant and mouse EB1 proteins have opposite intrinsic properties on the dynamic instability of microtubules.

“…Tubulin from different sources could in uence the functions of EB1 from plant and mice due to differences in tubulin composition and/or post-translational modi cations. Indeed, Arabidopsis presents a great diversity of alpha and beta tubulins and tubulin puri ed from tobacco possesses a different combination of post-translational modi cations than brain tubulin [18]. Although, interpretation was made easier having a reference point (mammalian EB1 on brain tubulin) to which we could compare the plant EB1, reproducing the work using plant tubulin would be bene cial.…”

Regulation of KIF1A motility via polyglutamylation of tubulin C-terminal tails

“…Tubulin was purified from HeLa Kyoto cells with TOG affinity column chromatography using gravity-flow setup [68,69]. Cells were resuspended in BRB80 (80 mM PIPES, 1 mM EGTA, 1 mM MgCl 2 pH 6.8) supplemented with 1 mM DTT and protease inhibitors (cOmpleteÔ, Mini, EDTA-free [Sigma-Aldrich, St. Louis, MO] and 0.2 mM PMSF) and sonicated.…”

“…Cleared lysate was loaded onto a TOG column, in which approximately 15 mg of bacterially purified GST-TOG1/2 protein was conjugated with 1 ml of NHS-activated Sepharose 4 Fast Flow resin (GE Healthcare; Marlborough, MA). Wash, elution, desalting and concentration was carried out as described in Hotta et al, 2016 [69]. Glycerol was not added to the purified tubulin.…”

Update: Plant Cortical Microtubule Arrays

“…The b-tubulin site is exposed, permitting GTP hydrolysis and exchange (Weisenberg et al, 1976). The functional relevance of different a/b-dimer combinations is still unknown but may soon be open for investigation with tools developed to purify specific tubulin isoforms and dimers with different posttranslational modifications from plant cells (Hotta et al, 2016). Tubulin modifications, such as C-terminal detyrosination (Duckett and Lloyd, 1994), phosphorylation, and acetylation, have long been known in plant cells and may confer functional effects in what has been dubbed a tubulin code (Cai, 2010;Xiong et al, 2013;Gadadhar et al, 2017;Takatani et al, 2017).…”

“…The kinetic bias for polymer accumulation likely has implications for events such as the rescue of newly severed plus ends and for the degree to which aging could affect the catastrophe frequency. Recent experiments with purified plant tubulin have indicated no special dynamic properties (Hotta et al, 2016), suggesting that the kinetic properties driving the steady-state treadmilling in plants are under the control of microtubule-associated proteins (MAPs) at the plus and minus ends.…”