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.

Microtubules (MTs) play important roles in biological functions by forming superstructures, such as doublets, triplets, and branched structures, in vivo. Formation of these superstructures by exogenous molecules in vitro will be useful not only for understanding the functions of MTs but also as components of MT-based nanomaterials. Here, we developed a tetrameric fluorescent protein Azami-Green (AG) fused with a His-tag and Tau-derived peptide (TP), TP-AG, which can bind to the inside or outside of MTs depending on the polymerization conditions. The binding of TP-AG to the inside of MTs induced the formation, stabilized, and increased the rigidity of the MTs. The binding of TP-AG to the outside of MTs induced various types of MT superstructures, including doublets, multiplets, and branched structures, by recruiting tubulins to MTs. The formation of motile MT aster structures by TP-AG was also observed. The generation of MT superstructures by these exogenous proteins provides guidelines for the design of MT-based nanomaterials.