Higher plant microtubule-associated proteins: In vitro functional assays

Abstract

Microtubule-associated proteins (MAPs) can account for the assembly and stabilization of microtubules at low tubulin concentration, for their ability to interact with other microtubules and/or cytoskeletal polymers or organelles and also for regulating microtubule anchoring and bundling properties. The data concerning higher plant MAPs remain limited so far to a few examples. Motor MAPs such as dynein or kinesin remain poorly documented in plants and are not to be discussed here. In this manuscript, the attention is focused on structural MAPs which co-assemble with tubulin during microtubule assembly. Using taxol, we developed an assay where higher plant microtubules were induced to self-assemble in a cytosolic extract of maize cultured cells and could be used as a native matrix for the isolation of putative higher plant MAPs. Seven polypeptides with molecular masses ranging between 60–125 kDa were found in this MAP-enriched fraction. These putative plant MAPs were shown to co-assemble with pig brain tubulin through two cycles of temperature-dependent assembly-disassembly. They were able to initiate and promote MAP-free tubulin assembly under conditions of non-efficient self-assembly and induced bundling of both plant and neural microtubules. One of these polypeptides (83 kDa) was found to be immunologically related to neural suggesting the presence of common epitopes between neural and plant MAPs. Such epitopes may be present at the microtubule-binding domains, as the higher plant MAPs co-assemble with brain tubulin. Plant microtubules exhibit an important in situ bundling activity, as in cortical or pre-prophase band arrays, or during the drastic reorganization of the cytoskeleton during mitosis induction. It is argued that different plant MAPs may regulate stability and function of these distinct microtubule populations. As higher plant cells lack defined centrosomes, specific MAPs could be involved in the control of the critical concentration of intracellular plant tubulin.