Formation of clathrin-pits and ATP-independent cholesterol-dependent tubules initiates mechano-regulation on de-adhesion.

Adherent cells ensure membrane homeostasis during de-adhesion by various mechanisms including endocytosis. Although mechano-chemical feedbacks involved in this process have been studied, the step-by-step build-up and resolution of the mechanical changes by endocytosis is not well understood. To investigate this, we study the de-adhesion of HeLa cells using a combination of interference reflection microscopy, optical-trapping and fluorescence experiments. We found that de-adhesion enhanced membrane height fluctuations of the basal membrane in the presence of an intact cortex. A reduction in the tether-force was also noted at the apical side. However, membrane fluctuations reveal phases of an initial drop in effective tension followed by a saturation. The area fractions of early (Rab5-labelled) and recycling (Rab4-labelled) endosomes as well as transferrin-labelled pits close to the basal plasma membrane also transiently increased. On blocking dynamin-dependent scission of endocytic pits, the regulation of fluctuations was not blocked but proceeded uncontrolled. Interestingly, the regulation could not be suppressed by ATP or cholesterol depletion individually but was arrested on depleting both. The data strongly supports pit-formation to be central to the reduction in fluctuations whether in normal or ATP depleted condition. Furthermore, while in normal conditions the contribution of clathrin-mediated endocytosis is clear, under ATP-depleted conditions we propose that cholesterol-dependent pits spontaneously regulate tension. SummaryWe show that during de-adhesion, cell edges retract, creating membrane folds and increasing fluctuations. Cells increase the rate of endocytosis to regulate back their membrane fluctuations. This is achieved by forming invaginations. Dynamin-dependent pathways are majorly involved, while cholesterol-dependent ATP-independent mechanisms also contribute.