In a study recently published in the journal Nano Letters, 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 (see fig. 1). The inner MT surface exhibited a corrugated structure, while the outer surface exhibited shallow undulations (see fig. 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 the 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.
