HeLa cells equilibrate membrane tension slowly, leading to inhomogeneities in plasma membrane mechanics. Yet how these mechanical heterogeneities connect to cellular function remains poorly understood. Here, we examine membrane tension distribution in cells and its relationship to endocytosis using tether force measurements, membrane fluctuation analysis, super-resolution imaging, and lipid compaction studies. While the basal tension was measured with interference reflection microscopy (IRM), apical tension measurements utilized optical trapping (OT). Using normal cells, mitotic cells, and plasma membrane spheres (PMS) lacking cytoskeleton, we show IRM and OT values to be correlated despite variability. However, sequential dual-tether experiments were essential to reveal a mid-plane peak and a vertical gradient, with tension decreasing with height at the apical surfaces. Disruption of actin inhibits this gradient, implicating actin in its maintenance. Super-resolution STED imaging of actin with myosin and ezrin confirmed that cytoskeletal contractility is involved. Flipper-TR lifetime measurements, which correlate with membrane tension, showed a sustained gradient in control cells that diminished after actin depletion and further decreased upon myosin II inhibition. PMS displayed minimal gradients, highlighting the role of actomyosin contractility in vertical tension gradient. Functionally, transferrin (Tf)-based endocytosis assays revealed that high-tension regions accumulated more transferrin at clathrin sites, increasing cargo-loaded pits. Inhibition of clathrin or dynamin steepened the Flipper-TR gradient, suggesting a balance between endocytosis, which reduces tension heterogeneity, and contractile forces, which preserve it. Together, our findings demonstrate that apical membrane tension is lower than at the midplane and that this vertical gradient is actively regulated by cytoskeletal contractility and endocytosis. This work reveals how cells maintain patterned membrane tension through coordinated cytoskeletal and functional mechanisms, uncovered by a correlative, multi-technique approach.
Ghosh et al. (Sun,) studied this question.