Higher actin contractility and cargo-loaded clathrin pits create midplane-peaked apico-basal tension profiles in HeLa cells

<h2>Summary</h2> Slow equilibration of local mechanical perturbations on the cell membrane could ideally create inhomogeneities in the mechanical state of the cell membrane, affecting its function. However, very few reports measure mechanical heterogeneities or connect them to cellular functional state. In this work, we employ optical-trap-based dual-tension measurements (per cell) to first reveal an actin-dependent tension gradient with low apical tensions. In the same cells, interference reflection microscopy reveals heterogeneous tension distribution at the base. Super-resolution imaging and fluorescence-lifetime (Flipper-TR) measurements reveal the midplane to be more contractile and tensed while confirming an actomyosin-contractility-dependent apical tension gradient and ruling out a direct contribution of actin-membrane linkages. Cargo-loaded clathrin-coated pits are bigger and denser at the tensed midplane. Inhibiting their formation suppresses the rise in midplane tension but accentuates the apical tension gradient. Thus, the tension profile is created by the actomyosin contractility profile but further tuned by its crosstalk with clathrin-mediated endocytosis.