Dissecting the cellular functions of plant microtubules using mutant tubulins

Abstract

a-and b-tubulins, the building blocks of the microtubule (MT) polymer, are encoded by multiple genes that are largely functionally redundant in plants. Null tubulin mutants are thus phenotypically indistinguishable from the wild type, but miss-sense or deletion mutations of critical amino acid residues that are important for the assembly, stability, or dynamics of the polymer disrupt the proper organization and function of the resultant MT arrays. Mutant tubulins co-assemble with wild-type tubulins into mutant MTs with compromised functions, and thus mechanistically act as dominant-negative MT poisons. Cortical MT arrays in interphase plant cells are most sensitive to tubulin mutations, and are transformed into helical structures or random orientation, which produce twisted or radially swollen cells. The second level of MT organization defines the overall morphology of the MT array. An example of this is the stable higher order arrangement of MTs found in the axoneme and basal body. In plant cells, MT arrays are rearranged during the progression of the cell cycle [Wasteneys, 2002]. As plant cells enter interphase, perinuclear MTs radiate from the periphery of the nucleus toward the plasma membrane. Soon after this stage, MTs become abundant at the cell periphery and associate closely with the plasma membrane. These cortical MTs interact with each other, form bundles, and become progressively aligned in parallel order. Bundled cortical MTs guide the movement of plasma membrane-localized cellulose synthase complexes, which synthesize load-bearing cellulose microfibrils, and ultimately instruct anisotropic growth, and hence cell shape [Bringmann et al., 2012]. At the end of the G2 phase, cortical arrays are transformed into prominent MT bundles, called the preprophase band, which usually encircle the nucleus at the cell cortex. The preprophase band is a transient MT structure that disappears at prophase, but specifies the eventual site of cell plate attachment at telophase. Breakdown of the nuclear envelope coincides with the formation of the mitotic

Microtubules in Plants

“…Most MT-interacting small molecules are derived from natural products, often of plant origin, and many of these are cells or tissues during development (Hashimoto, 2013a;Breviario et al, 2013). In Arabidopsis, TUA1 a-tubulin (At1g64740) and TUB9 b-tubulin (At4g20890) genes are exclusively or highly preferentially expressed in pollen (Carpenter et al 1992;Cheng et al, 2001).…”

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

“…Tubulins of Antarctic fishes accumulate unique amino acid substitutions at positions predicted to be important for MT dynamics and assemble into less dynamic and cold-stable MTs (Detrich et al, 2000). Even distinct tubulin isotypes in a given species show differences in dynamic instability (Hashimoto, 2013). In addition to the variability encoded in the tubulin genes, differences in posttranslational modifications between porcine brain tubulin and Arabidopsis tubulin (see below) might provide another layer of functional divergence in MT dynamics.…”

“…While the current method purifies single a-tubulin proteins, a similar strategy may be used to insert a purification tag into a poorly conserved luminal loop region of Arabidopsis b-tubulin. This approach would be useful for characterizing the effect of various tubulin mutations (Ishida et al, 2007;Hashimoto, 2013) and posttranslational modifications (see below) on MT dynamics and for analyzing the interactions of these mutated tubulins with MT regulators in vitro.…”

Natural Compounds as Next-Generation Herbicides

“…6;Hashimoto, 2013). Some of the most effective mitotic inhibitors discovered to date are natural products, such as colchicine from crocus (Colchicum spp.)…”