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.