Group led by Tottori University constructs microtubule superstructures using peptide-fused proteins

A research group led by Associate Professor Hiroshi Inaba and Professor Kazunori Matsuura of the Department of Engineering, Tottori University, in collaboration with a research group led by Assistant Professor Muneyoshi Ichikawa (currently at Fudan University) and Professor Tomoya Tsukazaki of the Division of Biological Science, Nara Institute of Science and Technology, and a research group led by Associate Professor Akira Kakugo and Professor Kazumi Sada of the Faculty of Science, Hokkaido University, has artificially constructed diverse superstructures composed of "microtubules," protein nanotubes that are a major component of the cytoskeleton, by using peptide-fused proteins they developed, for the first time in the world. Their research was published in Science Advances.

Microtubules generally take a singlet-type single winding structure, but in nature, they form a variety of superstructures, including doublet-type structures found in cilia and flagella and branched structures. However, these superstructures have been difficult to construct artificially.

By fusing a proprietary peptide that binds to the inner part of microtubules to a tetrameric fluorescent protein, the research group was able to control its binding to the inner and outer microtubules and induce the formation of various microtubule superstructures such as doublets, branches, microtubules of nearly 100 meters long, and asters, depending on the conditions.

They fabricated TP-AG by linking a Tau-derived peptide (TP), which binds to the tubulin pocket corresponding to the inner part of a microtubule, to the C-terminus of the tetrameric fluorescent protein AG (Azami-Green). They introduced a His tag at its N-terminus, which serves as a binding motif to the outer microtubule.

Since tubulin polymerizes to form microtubules by binding to guanosine triphosphate (GTP), the research group attempted to control the binding position to the microtubule by changing the timing of when TP-AG is added. In their first method, microtubules were constructed by complexing tubulin with TP-AG, and then adding GTP or its analogs. In their second method, TP-AG was complexed after the microtubules were fabricated.

In both methods, TP-AG was bound to the microtubules, but in the first method of complexing, TP-AG bound primarily to the inner part of the microtubules. In the second method, which was bound later, TP-AG was bound to the outer part of the microtubules. Microtubule formation was significantly augmented when TP-AG was complexed, forming stable microtubules. This augmentation and stabilization of microtubule formation was more effective than the anti-cancer drug Taxol, which functions by stabilizing microtubules. Furthermore, they found TP-AG to reinforce the microtubule structure, forming a more rigid structure than normal microtubules.

Negative staining electron microscopy of TP-AG complexed microtubules revealed doublet-shaped microtubules and branched structures not normally seen. The amount of those superstructures is particularly large in the way they are bound in the latter half, suggesting that the binding of TP-AG to the microtubule exterior induced the formation of superstructures. Cryo-EM analysis clearly showed the doublet structures induced by TP-AG to be A and B microtubules, and their diameters were almost identical to those of doublet microtubules isolated from cilia. Furthermore, the researchers successfully traced the growth of branched structures by using total reflection illumination fluorescence microscopy.

These results suggest that the binding of TP-AG to the outer microtubules induced the formation of doublets and branched structures by binding and accumulating new tubulin in the exposed TPs.

In addition to this, by changing conditions such as the amount of TP-AG and its complexation method, they succeeded in forming extremely long microtubule structures nearly 100 meters long and aster structures with motility. Thus, it became clear that despite the single type of peptide fusion protein, a variety of microtubule superstructures could be formed by changing the conditions.

This is the first example of microtubule superstructures formed by an exogenous protein and will greatly contribute to identifying the formation mechanism and physical properties of natural microtubule superstructures, which have been a mystery until now. Moving forward, this may lead to a better understanding of pathologies involving microtubule superstructures, such as ciliopathies. In addition, the doublet microtubules and branched structures obtained in this study have different structures and properties from those of ordinary microtubules, and thus have potential uses in nanomaterials such as molecular robots. They are anticipated to be applied to a wide range of fields, including the possible development into anti-cancer drugs using their extremely stabilized microtubule structure.

Journal Information
Publication: Science Advances
Title: Generation of stable microtubule superstructures by binding of peptide-fused tetrameric proteins to inside and outside
DOI: 10.1126/sciadv.abq3817