Studies on suppressors of sav2/shade avoidance 2 revealed altered interaction at the interface of αβ-tubulin intradimer affects microtubule dynamics

Abstract

Microtubules are highly dynamic cellular structures that are required for many biological processes. Cortical microtubules in plant play crucial roles during cell expansion. Its proper dynamics are required for plant growth and responses to environmental stimuli. Arabidopsis mutants, such as sav2/tub4P287L, display a variety of growth defects, including short and twisting hypocotyls in dark and shade. Both microtubule organization and dynamics are altered in sav2. Here, we have identified a suppressor of sav2 (sus2), which surprisingly contains a missense mutation in another β tubulin gene, TUB6. The mutation results in a L246F substitution in TUB6. It locates at the interface of αβ-intradimer. This mutation partially suppressed the swirling microtubule arrangement in sav2 hypocotyl cells, leading to the partial rescue of sav2 phenotypes. As the mutant behaves as a semi-dominant mutation and the CFP-labeled tub6L246F can incorporate into microtubules, we propose that the incorporation of tub6L246F interferes with the normal function of microtubules. tub6L246F single mutant is hypersensitive to drugs disrupting microtubule dynamics, such as colchicine, suggesting the mutation may affect microtubule dynamics. Moreover, we found the colchicine hypersensitivity of tub6L246F can be suppressed by tub4P287L, while tub6L246F interferes with the rescuing effect of EB1 on sav2. As P287 locates around M-loop, which is involved in interactions between microtubule protofilaments, we propose that altered interactions at αβ-intradimer interface may affect microtubule dynamics through M-loop mediated interactions between microtubule protofilaments.