title: Disruption of the actin cytoskeleton after microinjection of proteolytic fragments of alpha-actinin.
abstract: Alpha-actinin can be proteolytically cleaved into major fragments of 27 and 53 kD using the enzyme thermolysin. The 27-kD fragment contains an actin-binding site and we have recently shown that the 53-kD fragment binds to the cytoplasmic domain of beta 1 integrin in vitro (Otey, C. A., F. M. Pavalko, and K. Burridge. 1990. J. Cell Biol. 111:721-729). We have explored the behavior of the isolated 27- and 53-kD fragments of alpha-actinin after their microinjection into living cells. Consistent with its containing a binding site for actin, the 27-kD fragment was detected along stress fibers within 10-20 min after injection into rat embryo fibroblasts (REF-52). The 53-kD fragment of alpha-actinin, however, concentrated in focal adhesions of REF-52 cells 10-20 min after injection. The association of this fragment with focal adhesions in vivo is consistent with its interaction in vitro with the cytoplasmic domain of the beta 1 subunit of integrin, which was also localized at these sites. When cells were injected with greater than 5 microM final concentration of either alpha-actinin fragment and cultured for 30-60 min, most stress fibers were disassembled. At this time, however, many of the focal adhesions, particularly those around the cell periphery, remained after most stress fibers had gone. By 2 h after injection only a few small focal adhesions persisted, yet the cells remained spread. Identical results were obtained with other cell types including primary chick fibroblasts, BSC-1, MDCK, and gerbil fibroma cells. Stress fibers and focal adhesions reformed if cells were allowed to recover for 18 h after injection. These data suggest that introduction of the monomeric 27-kD fragment of alpha-actinin into cells may disrupt the actin cytoskeleton by interfering with the function of endogenous, intact alpha-actinin molecules along stress fibers. The 53-kD fragment may interfere with endogenous alpha-actinin function at focal adhesions or by displacing some other component that binds to the rod domain of alpha-actinin and that is needed to maintain stress fiber organization.
authors.affiliation: Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill 27599.
authors.forename: F M
authors.initials: FM
authors.lastname: Pavalko
authors.forename: K
authors.initials: K
authors.lastname: Burridge
mesh_terms: D000185:Actinin / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002642:Chick Embryo; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007150:Immunohistochemistry; D008845:Microinjections; D008856:Microscopy, Fluorescence; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D016596:Vinculin
chemical_list: D000199:Actins; D003598:Cytoskeletal Proteins; D010446:Peptide Fragments; D000185:Actinin; D016596:Vinculin