The spectrin skeleton of newly-invaginated plasma membrane

As a cell's shape and volume change, its surface area must re-adjust. How is the plasma membrane's spectrin skeleton implicated? For erythrocytes, cells of fixed surface area, spectrin responses to mechanical disturbances have been studied, but for more typical cells with changeable surface areas, they have not. In rapidly shrinking cells, surface membrane at an adherent substratum invaginates, forming transient vacuole-like dilations (VLDs). We exploited this readily inducible surface area perturbation to pose a simple question: is newly invaginated plasma membrane naked or is it supported by a spectrin skeleton? The spectrin skeleton was examined immunocytochemically in L6 cells (rat skeletal muscle) before and after VLD formation, using fixation in cold methanol and 4I12, an antibody against β-fodrin and β-spectrin. 4I12 was visualized by confocal fluorescence microscopy, while paired phase contrast images independently located the VLDs. To generate VLDs, cells were hypotonically swelled then reshrunk in isotonic medium. Swollen L6 cells maintained their plasma membrane (sarcolemma) spectrin skeleton. Within minutes of subsequent shrinkage, VLDs of 1–2 μm diameter invaginated at the substratum surface of myotubes. Both sarcolemma and VLDs were lined by a relatively uniform spectrin skeleton. Z-series suggested that some of the spectrin skeleton-lined sarcolemma became internalized as vacuoles.