Flexural Rigidity of Marginal Bands Isolated from Erythrocytes of the Newt

The marginal band is a bundle of microtubules residing at the periphery of nucleated erythrocytes of nonmammalian vertebrates and some invertebrates. Marginal bands from erythrocytes of the newt (Notopthalmus viridescens) were isolated from the cells as intact structures by treatment with detergent and either mild protease or high salt. Isolated bands were subjected to mechanical testing by stretching the band between a glass microhook and a calibrated glass fiber. The deflection of the fiber provided a measure of the force on the band. The flexural rigidity of the band was determined from measurements of the band deformation as a function of applied force. Bands isolated with either of two proteases (pepsin or elastase) or with high salt exhibited elastic behavior with a flexural rigidity of ~9.0 x 10 -~2 dyn-cm 2. Treatment of bands with chymopapain caused an increase in band rigidity and inelastic behavior. Estimates of the contribution of the band to cellular rigidity are made based on the measurements of the structural properties of the isolated band. The band provides the cell with a large resistance to indentations at the rim and to large extensions, while maintaining a high degree of flexibility in small extensions or flexure. T n~ marginal band is a major cytoskeletal element in erythrocytes of nonmammalian vertebrates, in erythrocytes of some invertebrates, and in mammalian platelets. It consists of a bundle of microtubules located at the periphery of the cell just beneath the plasma membrane. Its function appears to be mechanical. It has been implicated in the determination of cell shape during morphogenesis (1) and in stabilizing the shape of the mature cell (10). Unlike other microtubular structures, some marginal bands remain stable after cell lysis and, under proper conditions, they can be isolated intact from the rest of the cytoskeleton. This feature makes them an attractive model system for studying the structure, composition, and mechanical properties of a microtubular assembly. Evidence that the marginal band plays a role in stabilizing the shape of the mature erythrocyte was first obtained by Joseph-Silverstein and Cohen (10). They observed that cells subjected to mechanical stresses are more grossly deformed when the marginal band is absent than when it is present. Because marginal bands can be isolated as intact structures it is possible to study their properties directly and quantitatively. In a previous report from this laboratory (17), a method was described for determining the resistance of the band to flexure (a change in curvature) and extension (a change in the length of its contour). These structural properties are characterized by two coefficients: the flexural rigidity (El) and the extensional rigidity (EA). In that previous study accurate determinations of the extensional rigidity were made, but, because the applied forces were large, the determination of the flexural rigidity was only approximate. In the present study we apply smaller forces to isolated bands to make accurate measurements of the flexural rigidity. To control against possible artifactual effects of the isolation procedure on the structural properties of the band, we have tested bands isolated by four different methods. Three of the methods, one using pepsin, one using elastase, and one using high salt in the presence of protease inhibitors, produced bands with nearly identical properties. Treatment of the bands with chymopapain was found to produce a large increase in band rigidity. Materials and Methods