Characteristics of a membrane reservoir buffering membrane tension

When membrane-attached beads are pulled vertically by a laser tweezers, a m embrane tube of constant diameter (tether) is formed. We found that the for ce on the bead (tether force) did not depend on tether length over a wide r ange of tether lengths, which indicates that a previously unidentified rese rvoir of membrane and not stretch of the plasma membrane provides the tethe r membrane. Plots of tether force vs. tether length have an initial phase, an elongation phase, and an exponential phase. During the major elongation phase, tether force is constant, buffered by the "membrane reservoir." Fina lly, there is an abrupt exponential rise in force that brings the tether ou t of the trap, indicating depletion of the membrane reservoir. In chick emb ryo fibroblasts and 3T3 fibroblasts, the maximum tether lengths that can be pulled at a velocity of 4 mu m/s are 5.1 +/- 0.3 and 5.0 +/- 0.2 mu m, res pectively. To examine the importance of the actin cytoskeleton, we treated cells with cytochalasin B or D and found that the tether lengths increased dramatically to 13.8 +/- 0.8 and 12.0 +/- 0.7 mu m, respectively. Similarly , treatment of the cells with colchicine and nocodazole results in more tha n a twofold increase in tether length. We found that elevation of membrane tension (through osmotic pressure, a long-term elevation of tether force, o r a number of transitory increases) increased reservoir size over the whole cell. Using a tracking system to hold tether force on the bead constant ne ar its maximal length in the exponential phase, the rate of elongation of t he tethers was measured as a function of tether force (membrane tension). T he rate of elongation of tethers was linearly dependent on the tether force and reflected an increase in size of the reservoir. Increases in the reser voir caused by tension increases on one side of the cell caused increases i n reservoir size on the other side of the cell. Thus, we suggest that cells maintain a plasma membrane reservoir to buffer against changes in membrane tension and that the reservoir is increased with membrane tension or disru ption of the cytoskeleton.