Atomic force microscopy reveals microtubule defects with submolecular resolution

In a study recently published in the journal Nano Letters, researchers at Kanazawa University Nano Life Science Institute (WPI-NanoLSI) used frequency-modulated atomic force microscopy to examine the inner surfaces of microtubules (MTs) and microtubules. We revealed the submolecular structure of the internal surface of (MT). We visualize structural defects in the MT lattice and provide valuable insight into the complex dynamic processes that regulate microtubule function.

Microtubules (MTs), an important component of the eukaryotic cell cytoskeleton, serve as scaffolds and play important roles in cellular processes such as cell division, cell migration, intracellular trafficking, and transport. MTs are composed of α- and β-tubulin proteins, which polymerize into dimers and assemble into linear protofilaments that form a cylindrical lattice.

Traditional methods such as X-ray crystallography and cryo-electron microscopy have provided insight into the structure of microtubules, but require complex sample preparation and data analysis. There remains a need for techniques that can examine the structural features, assembly dynamics, and lattice defects of MTs at submolecular resolution under physiological conditions.

The external surface of MT walls has been extensively studied. However, there are limited studies investigating the intramolecular arrangement of tubulin dimers within the MT wall. The outer and inner walls of microtubules interact with a variety of proteins.

To address this gap, a team of scientists led by Ayhan Yurtseva, Hitoshi Asakawa, and Takeshi Fukuma from Kanazawa University NanoLife Science Institute (WPI-NanoLSI) developed a frequency-modulated atomic force microscope (FM-AFM). was employed to study submolecular substances. Tubulin dimers are located on both the inner and outer surfaces of MTs (see Figure 1). The inner surface of the MT exhibited a corrugated structure, whereas the outer surface exhibited shallow relief (see Figure 2).

One protofilament was topographically higher than its neighboring protofilament. This different topography was caused by differences in the structural orientation and conformation of αβ-tubulin heterodimers between adjacent protofilaments. The protofilament α-tubulin and β-tubulin monomers on the inner surface are reoriented during the tube-to-sheet conformational transition.

There is also a “seam” line on the inner surface, which is thought to give flexibility to the MT. FM-AFM enabled the detection of several lattice defects or structural defects caused by the loss of tubulin subunits along the protofilaments of MT lattice shafts in local regions. These defects can alter the molecular arrangement of protofilaments, thereby impairing microtubule function.

This study also investigated the interaction of MTs with taxol, a chemotherapeutic drug that binds exclusively to the β-tubulin subunit within αβ-tubulin dimers on the internal surface of MTs. Taxol-stabilized microtubules may inhibit cancer cell division and migration, thereby slowing cancer progression.

This binding served as a marker to distinguish between individual α and β-tubulin subunits in high-resolution AFM images. This insight highlights the potential of FM-AFM to study the molecular mechanisms of microtubule-targeted drugs.

In summary, FM-AFM provides important insights into MT structure, dynamics, and drug interactions, revealing potential to advance drug discovery. Understanding microtubule function and protein interactions may help develop more specific and efficient therapies, especially for cancers where microtubules are a major therapeutic target.