Exploring microtubule lattice at submolecular resolution

In a study recently published in the journal Nano Letters, published by American Chemical Society, researchers from Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kanazawa, Japan, used frequency-modulated atomic force microscopy to reveal the submolecular structure of microtubule (MT) inner surface and visualize structural defects in the MT lattice, providing valuable insights into the complex dynamic processes that regulate microtubule function.

Microtubules (MTs), a key component of the cytoskeleton in eukaryotic cells, serve as scaffolds and play vital roles in cellular processes such as cell division, cell migration, intracellular transport, and trafficking. MTs are composed of α-tubulin and β-tubulin proteins, which polymerize into dimers and assemble into linear protofilaments that form a cylindrical lattice. Traditional methods like X-ray crystallography and cryo-electron microscopy have provided structural insights into MTs but involve complex sample preparation and data analysis. There remains a need for techniques that can examine MT structural features, assembly dynamics, and lattice defects at submolecular resolution under physiological conditions.

The outer surface of the MT wall has been extensively studied. However, limited studies have examined the submolecular arrangement of tubulin dimers in the inner MT wall. The outer and inner walls of MTs interact with different proteins. To address this gap, a team of scientists led by Ayhan Yurtsever, Hitoshi Asakawa, and Takeshi Fukuma at Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, employed frequency-modulation atomic force microscopy (FM-AFM) to study the submolecular arrangement of tubulin dimers on both the inner and outer MT surfaces (Figure 1). The inner MT surface exhibited a corrugated structure, while the outer surface exhibited shallow undulations (Figure 2). One protofilament was topographically higher than its adjacent protofilament. This differential topography was caused by differences in the structural orientations and conformation of αβ-tubulin heterodimers between adjacent protofilaments. The α-tubulin and β-tubulin monomers of the protofilaments on the inner surface reoriented during the structural transition from tubes to sheets. The inner surface also had a “seam” line, which is considered to confer flexibility to MTs. FM-AFM enabled the detection of several lattice or structural defects caused by missing tubulin subunits along the protofilaments in the MT lattice shaft in the localized region. These defects can alter the molecular arrangement of protofilaments and consequently impair the functions of MTs.

The study also explored MT interactions with Taxol, a chemotherapy drug that exclusively binds to β-tubulin subunits within αβ-tubulin dimers on the inner MT surface. Taxol-stabilized microtubules inhibit cancer cell division and migration, thereby potentially slowing cancer progression. This binding served as a marker to distinguish individual α- and β-tubulin subunits in high-resolution AFM images. This insight underscores FM-AFM's potential to investigate the molecular mechanisms of drugs that target MTs.

In summary, FM-AFM provides critical insights into MT structure, dynamics, and drug interactions, revealing potential for advancing drug discovery. Understanding MT function and protein interactions can guide the development of more specific and efficient therapies, particularly for cancer, where MTs are key therapeutic targets.

Funding Information

This work was primarily supported by Grants in Aid for Scientific Research (21H05251 and No. 20K05321) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. This work was also partially supported by the World Premier International Research Center Initiative (WPI), MEXT, Japan

About Nano Life Science Institute (WPI-NanoLSI), Kanazawa University

Understanding nanoscale mechanisms of life phenomena by exploring “uncharted nano-realms”.

Cells are the basic units of almost all life forms. We are developing nanoprobe technologies that allow direct imaging, analysis, and manipulation of the behavior and dynamics of important macromolecules in living organisms, such as proteins and nucleic acids, at the surface and interior of cells. We aim at acquiring a fundamental understanding of the various life phenomena at the nanoscale.

https://nanolsi.kanazawa-u.ac.jp/en/

About the World Premier International Research Center Initiative (WPI)

The WPI program was launched in 2007 by Japan's Ministry of Education, Culture, Sports, Science and Technology (MEXT) to foster globally visible research centers boasting the highest standards and outstanding research environments. Numbering more than a dozen and operating at institutions throughout the country, these centers are given a high degree of autonomy, allowing them to engage in innovative modes of management and research. The program is administered by the Japan Society for the Promotion of Science (JSPS).

See the latest research news from the centers at the WPI News Portal: https://www.eurekalert.org/newsportal/WPI

Main WPI program site: www.jsps.go.jp/english/e-toplevel

About Kanazawa University

As the leading comprehensive university on the Sea of Japan coast, Kanazawa University has contributed greatly to higher education and academic research in Japan since it was founded in 1949. The University has three colleges and 17 schools offering courses in subjects that include medicine, computer engineering, and humanities.

The University is located on the coast of the Sea of Japan in Kanazawa – a city rich in history and culture. The city of Kanazawa has a highly respected intellectual profile since the time of the fiefdom (1598-1867). Kanazawa University is divided into two main campuses: Kakuma and Takaramachi for its approximately 10,200 students including 600 from overseas.

http://www.kanazawa-u.ac.jp/en/