Microvilli are membrane protrusions involved in many membrane-associated physiological processes. Previous studies have focused on the dynamics of individual microvilli, however, the morphological classification of microvilli and the dynamics of microvillar clusters as the basic functional domain remain largely unknown. Here we used atomic force microscopy (AFM) to achieve nanoscale resolution 3D microvilli images of living HeLa cells. We found that there were mainly two subtypes of microvilli with different morphologies and lifecycle that were unequally present on the cell membrane. Employing a strategy to track microvillar cluster movement at nanometer resolution, we further revealed the polymorphic movement of microvillar clusters in 3D. Overall, these data strengthened the morphology and dynamics of cell membranes and associated structures, which provided a new perspective for microvillar function research.
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