Membrane blebbing is associated with Ca²⁺‐activated hyperpolarizations induced by serum and α₂‐macroglobulin

We have reported previously that serum and α₂‐macroglobulin (α₂M) induce Ca²⁺‐activated hyperpolarizations in the membrane potential of a clonal rat osteosarcoma cell line (ROS 17/2) (Dixon and Aubin, J. Cell. Physiol., 132:215–225, 1987). In this report, we describe morphological changes that accompany these hyperpolarizations. Both cell surface blebbing (zeiosis) and transient hyperpolarizations were induced by application of 10% fetal bovine serum (FBS) or α₂M; neither was induced by serum‐free medium, a suspension of latex beads, or purified bovine serum albumin. Following a brief application of FBS or α₂M at time 0, electrical activity typically occurred between 7–40 s and was always followed by blebbing activity that began at 30 s and persisted for 3–5 min. In contrast, continuous exposure to FBS resulted in the persistence of both blebbing activity and transient hyperpolarizations for periods of at least several hours. Scanning electron microscopy (SEM) revealed that the blebs appeared concomitantly with the disappearance of microvilli and the appearance of surface pits that measured 100–300 nm in diameter. Coated pits and vesicles, similar in size to the pits observed by SEM, were observed using transmission electron microscopy (TEM). By TEM, blebs were found to contain few organelles other than centrally located free ribosomes. Fluorescence microscopy of nitrobenzooxadizole‐phallacidin‐labeled cells indicated that blebs contained filamentous actin and that microfilament bundles remained primarily on the substratum side of blebbed cells. We propose that blebbing results from a dynamic local reorganization of microfilaments initiated by ligand‐induced transient increases in intracellular Ca²⁺.