Non-erythrocyte spectrin network preferentially stabilizes flat membrane and enhances cell stiffness

Spectrin αII and βII, the predominant non-erythroid isoforms, assemble into cytoskeletal networks that shape the plasma membrane. However, how these networks interact with membranes of different curvatures remains unclear. Using microfluidic deformation cytometry, we show that spectrin βII overexpression increases the apparent stiffness of MDA-MB-231 breast cancer cells. Fluorescence microscopy further demonstrates that spectrin is excluded from highly curved regions and enriched on flatter membranes <i>in vivo</i>, specifically those with an absolute curvature |<i>κ</i>| < 0.2 µm^-1. Consistently, <i>in vitro</i> reconstitution with spherical supported lipid bilayers (SSLBs) shows that purified spectrin heterodimers preferentially bind low-curvature membranes, exhibiting ∼15-fold higher association with 1000 nm SSLBs (|<i>κ</i>| ≈ 0.5 µm^-1) than with 30 nm SSLBs (|<i>κ</i>| ≈ 66.7 µm^-1). This common curvature-dependent preference is promoted by spectrin oligomerization. Together, these results establish spectrin as a curvature-responsive cortical scaffold that selectively stabilizes flat membrane domains, thereby maintaining cellular stiffness.