Cyclic AMP-induced shape changes of astrocytes are accompanied by rapid depolymerization of actin

Abstract

Agents that increase intracellular cyclic AMP produce a process-bearing morphology in astrocytes. We have examined short-term re-arrangements of the astrocyte cytoskeleton during this shape conversion. Primary cultures of astrocytes from neonatal rat forebrain were grown at low density as polygonal shaped cells. Treatment with 1 mM dibutyryl cAMP in the absence of serum produced rapid changes in cell shape (100% of cells as flat to 90% showing cytoplasmic contraction and processes in 60 min). In the presence of serum, similar changes took place, but more slowly. No changes in total cellular levels of GFAP, vimentin, tubulin or actin were observed over a 2-h period of treatment. There was a shift in actin from a Triton X-100-insoluble pool to a soluble pool, with a 40% reduction in insoluble actin. The kinetics of this shift paralleled kinetics of shape change. The shift also corresponded to a loss of stress fibers, visualized with rhodamine-phalloidin. Intermediate stages of stress fiber loss were observed as short, wavy or small ring profiles. Colchicine prevented the dBcAMP-induced changes in shape. If cells were first treated with taxol, however, subsequent exposure to colchicine did not inhibit contraction. Thus, dBcAMP, presumably through a cAMP-dependent kinase, depolymerizes actin in stress fiber forms as cells contract. In addition, an intact microtubule system may be required for the changes in shape. Treatment with dBcAMP also caused the disappearance of vinculin-containing attachment sites, indicating that adhesion plaques, or at least the association of vinculin with them, are lost during the time of microfilament bundle dissociation.