Tubulin isotype substitution revealed that isotype combination modulates microtubule dynamics in C. elegans embryos

Abstract

Microtubules (MTs) are polymers composed of α-and β-tubulin heterodimers that are generally encoded by genes at multiple loci. Despite implications of distinct properties depending on the isotype, how these heterodimers contribute to the diverse MT dynamics in vivo remains unclear. Here, by using genome editing and depletion of tubulin isotypes following RNAi, we demonstrate that four tubulin isotypes (hereafter referred to as α1, α2, β1 and β2) cooperatively confer distinct MT properties in Caenorhabditis elegans early embryos. GFP insertion into each isotype locus reveals their distinct expression levels and MT incorporation rates. Substitution of isotype coding regions demonstrates that, under the same isotype concentration, MTs composed of β1 have higher switching frequency between growth and shrinkage compared with MTs composed of β2. Lower concentration of β-tubulins results in slower growth rates, and the two α-tubulins distinctively affect growth rates of MTs composed of β1. Alteration of ratio and concentration of isotypes distinctively modulates both growth rate and switching frequency, and affects the amplitude of mitotic spindle oscillation. Collectively, our findings demonstrate that MT dynamics are modulated by the combination (ratio and concentration) of tubulin isotypes with distinct properties, which contributes to create diverse MT behaviors in vivo.

CRISPR/Cas-mediated knock-in via non-homologous end-joining in the crustacean Daphnia magna

“…The Dma-ey gene was chosen as a target of Cas9 RNPs. For the preparation of the Dma-ey -targeting Cas9 RNPs, we purified Cas9 proteins as described previously [ 34 ] and used two different Dma-ey gRNAs named gRNA-1 and gRNA-2, which had been used previously in knock-out experiments using Cas9 mRNAs ( Fig 1 ) [ 25 ]. The gRNA-1 and gRNA-2 sequences target exons 8 and 10 of the Dma-ey gene, respectively ( Fig 1 ).…”

Human β-Tubulin Isotypes Can Regulate Microtubule Protofilament Number and Stability

“…Their study shows that C. elegans tubulin, purified as a mixture of isotypes, can form microtubules with fewer protofilaments than the canonical 13, indicating that the intrinsic properties of tubulin contribute to the smaller protofilament numbers observed for microtubules in embryos as well as in other cell types in the adult animal (Chalfie and Thomson, 1982). Interestingly, the purified tubulin has the unusually fast microtubule growth rates observed in C. elegans embryos (Honda et al, 2017; Srayko et al, 2005). The authors provide evidence that these rapid polymerization rates are likely due to the H1-S2 and M loops in C. elegans tubulin adopting secondary structure more readily than other slower polymerizing tubulins, effectively pre-paying the entropic costs that limit polymerization rates.…”

The coordination of spindle-positioning forces during the asymmetric division of theC. eleganszygote is revealed by distinct microtubule dynamics at the cortex

“…We next asked whether the dynamical behaviours of the tracks reflect the presence of two populations of microtubules and investigated possible artefacts caused firstly by the microtubule labelling. Indeed, it was recently proposed that tubulin paralogs composition of the microtubules impacted their dynamics (Honda et al, 2017) or the spindle positioning (Wright and Hunter, 2003) in the C. elegans one-cell embryo. Thus, we asked whether YFP::α-tubulin fluorescent labelling could create two populations.…”