Visualizing\nthe Submolecular Organization of αβ-Tubulin\nSubunits on the Microtubule Inner Surface Using Atomic Force Microscopy

Microtubules (MTs) are dynamic cytoskeletal polymers\nessential\nfor mediating fundamental cellular processes, including cell division,\nintracellular transport, and cell shape maintenance. Understanding\nthe arrangement of tubulin heterodimers within MTs is key to their\nfunction. Using frequency modulation atomic force microscopy (FM-AFM)\nand simulations, we revealed the submolecular arrangement of α-\nand β-tubulin subunits on the inner MT surface. We observed\nan undulating molecular arrangement of protofilaments (PFs) with alternating\nheight variations, attributed to different structural orientations\nand the confirmation of αβ-tubulin heterodimers in adjacent\nPFs, forming bimodal lateral contacts, as confirmed by AFM simulations.\nStructural defects resulting from missing tubulin units were directly\nidentified. This detailed structural information provides critical\ninsight into the MT functional properties. Our findings highlight\nthe potential of FM-AFM in liquid as a powerful tool for elucidating\nthe complex interactions among MTs, MT-associated proteins, and other\nmolecules, which are essential for understanding MT dynamics in the\ncellular context.