Generation of stable microtubule superstructures by binding of peptide-fused tetrameric proteins to inside and outside

Abstract

Microtubules play important roles in biological functions by forming superstructures, such as doublets and branched structures, in vivo. Despite the importance, it is challenging to construct these superstructures in vitro. Here, we designed a tetrameric fluorescent protein Azami-Green (AG) fused with His-tag and Tau-derived peptide (TP), TP-AG, to generate the superstructures. Main binding sites of TP-AG can be controlled to the inside and outside of microtubules by changing the polymerization conditions. The binding of TP-AG to the inside promoted microtubule formation and generated rigid and stable microtubules. The binding of TP-AG to the outside induced various microtubule superstructures, including doublets, multiplets, branched structures, and extremely long microtubules by recruiting tubulins to microtubules. Motile microtubule aster structures were also constructed by TP-AG. The generation of various microtubule superstructures by a single type of exogenous protein is a new concept for understanding the functions of microtubules and constructing microtubule-based nanomaterials.

Assembly properties ofSpiroplasmaMreB involved in swimming motility

“…5B), suggesting that non-specific interactions via the C-terminal region increase the local concentration of SpeMreB5 around sheets and paracrystals to promote their assemblies. This idea is consistent with a previous study in which engineered proteins with flexible tubulin-binding regions on the outside of microtubules induced supra-structural formations such as microtubule doublets and branched microtubules (37). As the C-terminal region of SciMreB5 is involved in binding to the negatively charged Spiroplasma membrane (3), the paracrystal nucleation property may induce MreB5 nucleation on the membrane.…”

Native doublet microtubules from Tetrahymena thermophila reveal the importance of outer junction proteins

“…Therefore, it is likely that CFAP77 contributes more to the stability than the assembly of the outer junction. It was recently shown that Azami Green-fused Tau-derived peptide added to the microtubules induces formation of the more complex structures, including DMT 42 similar to the doublet reconstitution of C-terminal tail truncated tubulins 22 . Therefore, it’s possible that other proteins in primary cilia bind to the outer surface of microtubules and interact with or suppress the C-terminal tails of tubulins.…”

Native doublet microtubules fromTetrahymena thermophilareveal the importance of outer junction proteins

“…Therefore, it is likely that in non-motile primary cilia the DMT assembly is CFAP77-independent. It was recently shown that Azami Green-fused Tau-derived peptide added to the microtubules induces formation of the more complex structures, including DMT (Inaba et al, 2022) similar to the doublet reconstitution of C-terminal tail truncated tubulins (Schmidt-Cernohorska et al, 2019). Therefore, it’s possible that other proteins in primary cilia bind to the outer surface of microtubules and interact with or suppress the C-terminal tails of tubulins.…”