A decrease in membrane tension precedes successful cell-membrane repair

We hypothesized that the requirement for Ca2+-dependent exocytosis in tell-membrane repair is to provide an adequate lowering of membrane tension to permit membrane resealing. We used laser tweezers to form membrane tethers and measured the force of those tethers to estimate the membrane tension of Swiss 3T3 fibroblasts after membrane disruption and during resealing. These measurements show that, for fibroblasts wounded in normal Ca2+ Ringer's solution, the membrane tension decreased dramatically after the wounding and resealing coincided with a decrease of similar to 60% of control tether force values. However, the tension did not decrease if cells were wounded in a low Ca2+ Ringer's solution that inhibited both membrane resealing and exocytosis. When cells were wounded twice in normal Ca2+ Ringer's solution, deceases in tension at the second wound were 2.3 times faster than at the first wound, correlating well with twofold faster resealing rates for repeated wounds. The facilitated resealing to a second wound requires a new vesicle pool, which is generated via a protein kinase C (PKC)-dependent and brefeldin A (BFA)-sensitive process. Tension decrease at the second wound was slowed or inhibited by PKC inhibitor or BFA. Lowering membrane tension by cytochalasin D treatment could substitute for exocytosis and could restore membrane resealing in low Ca2+ Ringer's solution.