?-Tubulin 2 nucleates microtubules and is downregulated in mouse early embryogenesis.

ABSTRACT: ?-Tubulin is the key protein for microtubule nucleation. Duplication of the ?-tubulin gene occurred several times during evolution, and in mammals ?-tubulin genes encode proteins which share ?97% sequence identity. Previous analysis of Tubg1 and Tubg2 knock-out mice has suggested that ?-tubulins are not functionally equivalent. Tubg1 knock-out mice died at the blastocyst stage, whereas Tubg2 knock-out mice developed normally and were fertile. It was proposed that ?-tubulin 1 represents ubiquitous ?-tubulin, while ?-tubulin 2 may have some specific functions and cannot substitute for ?-tubulin 1 deficiency in blastocysts. The molecular basis of the suggested functional difference between ?-tubulins remains unknown. Here we show that exogenous ?-tubulin 2 is targeted to centrosomes and interacts with ?-tubulin complex proteins 2 and 4. Depletion of ?-tubulin 1 by RNAi in U2OS cells causes impaired microtubule nucleation and metaphase arrest. Wild-type phenotype in ?-tubulin 1-depleted cells is restored by expression of exogenous mouse or human ?-tubulin 2. Further, we show at both mRNA and protein levels using RT-qPCR and 2D-PAGE, respectively, that in contrast to Tubg1, the Tubg2 expression is dramatically reduced in mouse blastocysts. This indicates that ?-tubulin 2 cannot rescue ?-tubulin 1 deficiency in knock-out blastocysts, owing to its very low amount. The combined data suggest that ?-tubulin 2 is able to nucleate microtubules and substitute for ?-tubulin 1. We propose that mammalian ?-tubulins are functionally redundant with respect to the nucleation activity.

SUBMITTER: Vinopal S 

PROVIDER: S-EPMC3250491 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature