Microtubules are hollow cytoskeletons with a typical 15 nm inner diameter, comprising tubulin proteins. Due to their intriguing properties such as high stiffness, reversible formation/dissociation, motility combined with motor proteins, and the ability to form superstructures such as doublets and branches, microtubules are vital cellular components and interesting building blocks for nanomaterials. Engineering the structures and functions of microtubules is pivotal for understanding their biological significance and exploring nanotechnological applications. While the “outside” of microtubules has been widely used for molecular conjugation, the “inside” of microtubules has not been focused. We have previously developed a Tau-derived peptide (TP) as a binding unit to the inside of microtubules (Chem. Eur. J., 2018, 24, 14958) and used TP for encapsulating various nanomaterials in microtubules (Account: Bull. Chem. Soc. Jpn., 2021, 94, 2100).

Herein, we discuss our microtubule engineering approach with TP-fused proteins. First, TP-fused GFP (TP-GFP) was constructed and encapsulated in microtubules (Chem. Commun., 2019, 55, 9072). The encapsulation of TP-GFP resulted in structural changes in the microtubules, an increase in contour length, persistence length, and velocity driven by ATP on the kinesin-coated plate. TP-GFP also enhanced tubulin polymerization like taxol, an anticancer drug that stabilizes microtubules. Next, TP was fused to Azami-Green (AG), a protein that generates fluorescence by tetramerization, for the generation of microtubule superstructures (Sci. Adv., 2022, 8, eabq3817). Binding sites of TP-fused AG (TP-AG) to microtubules were controlled by changing the incorporation methods. Binding of TP-AG to the inside induced dramatic stabilization of microtubules, which was superior to the effects of TP-GFP and taxol. Binding of TP-AG to the outside induced the formation of various microtubule superstructures such as doublets and branches. ATP-driven motile aster structures were also observed by using TP-AG-incorporated microtubules. These results indicate that the binding of exogenous proteins to microtubules can effectively engineer microtubules.