A Decrease in Membrane Tension Precedes Successful Cell-Membrane Repair
Abstract
We hypothesized that the requirement for Ca-dependent exocytosis in cell-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 Ca Ringer's solution, the membrane tension decreased dramatically after the wounding and resealing coincided with a decrease of ∼60% of control tether force values. However, the tension did not decrease if cells were wounded in a low Ca Ringer's solution that inhibited both membrane resealing and exocytosis. When cells were wounded twice in normal Ca Ringer's solution, decreases 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 Ca Ringer's solution.
Cells were treated with DMSO or 20 μM cytochalasin D for 1 h and wounded. Membrane sealing was monitored by photometric measurement of fura-2 fluorescence and visual inspection (see MATERIALS AND METHODS).
ACKNOWLEDGMENTS
We thank Bruce J. Tromberg for providing access to the facilities of the Beckman Laser Institute and for insightful suggestions. This study was supported by the National Institutes of Health (R01AR44066 and P41RR01192). We also thank William and Patricia Baker of Corona Del Mar, CA, who generously opened their home to us to stay there during our work.