Regulation of plasma membrane blebbing by the cytoskeleton

When neuroblastoma cells are exposed to lysophosphatidic acid (LPA), they u ndergo a vigorous, but transient blebbing phase. The effect is sensitive to inhibition by staurosporine, KT 5926 (an inhibitor of myosin light chain k inase), and cytochalasin B, suggesting that LPA activates the phosphorylati on of myosin light chain and increases the contractile activity of the acto myosin network. Cell contractions increase the intracellular pressure drivi ng bleb formation. Calyculin, an inhibitor of protein phosphatase2A, also c auses blebbing which continues as long as the drug is present, presumably b y keeping myosin light chain in the phosphorylated state. Blebbing of neuro blastoma cells is regulated by the status of all three cytoskeletal systems : disassembly of microtubules by nocodazole and of intermediate filaments b y acrylamide increased the number of blebbing cells. Cytochalasin B, on the other hand, prevents bleb retraction and, after prolonged incubation, bleb formation. These results are discussed in terms of a model viewing the cyt oskeleton as an integrated network transmitting force throughout the cell. Bleb retraction was studied by transfecting neuroblastoma cells with a vect or containing the gene for gamma-cytoplasmic actin fused to the green fluor escent protein EGFP (EGFP-actin). EGFP-actin was not detected on the membra nes of extending blebs, but started accumulating along the cytoplasmic surf ace of blebs as soon as the extension phase came to an end and retraction s et in. These results confirm earlier suggestions that actin polymerization is required for bleb retraction and for the first time directly relate the two events. (C) 1999 Wiley-Liss, Inc.