Disruption ofthe Actin Cytoskeleton after Microinjection of Proteolytic Fragments of aActinin

a-Actinin can be proteolytically cleaved into major fragments of 27 and 53 kD using the enzyme thermolysin . The 27-kD fragment contains an actinbinding site and we have recently shown that the 53kD fragment binds to the cytoplasmic domain of a, 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 27and 53-kD fragments of a-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 a-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 al subunit of integrin, which was also localized at these sites. When cells were injected with >5 uM final concentration of either a-ACTININ is an actin binding protein that cross-links actin filaments in vitro (Maruyama and Ebashi, 1965 ; Burridge and Feramisco, 1981) . Its native structure is a homodimer with a subunit molecular weight of 103 kD (Singh et al ., 1977; Suzuki et al ., 1979 ; Baron et al ., 1987) and is visualized in the electron microscope as a dumbbell-shaped molecule, 30-40 tun long and 3-4 nm wide with an enlarged head domain at each end (Podlubnaya et al ., 1975 ; Bretscher et al., 1979 ; Pollard, 1981) . Distinct a-actinin isoforms have been identified in muscle (Endo and Masaki, 1982) and nonmuscle cells (Burridge and Feramisco, 1981 ; Duhaiman and Bamburg, 1984 ; Bennett et al ., 1984 ; Landon et al ., 1985) ; however, the only known functional difference between the isoforms is that binding of muscle a-actinin to actin is calcium insensitive, whereas the binding of nonmuscle a-actinins to actin is inhibited by calcium (Mimura and Asano, 1979; Burridge and Feramisco, 1981 ; Duhaiman and Bamburg, 1984 ; Bennet et al ., 1984 ; Landon et al ., 1985) . Based on the complete amino acid sequence of a-actinin from various sources (Baron et al ., 1987 ; Noegel et al ., F. M. Pavalkds current address is Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis, IN 45202 . © The Rockefeller University Press, 0021-9525/91/08/481/11 $2 .00 The Journal of Cell Biology, Volume 114, Number 3, August 1991481-491 a-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 a-actinin into cells may disrupt the actin cytoskeleton by interfering with the function of endogenous, intact «-actinin molecules along stress fibers . The 53-kD fragment may interfere with endogenous a-actinin function at focal adhesions or by displacing some other component that binds to the rod domain of a-actinin and that is needed to maintain stress fiber organization . 1987) it has become clear that the a-actinin monomer is organized into three domains : a globular NHZ-terminal domain which contains the binding site for actin (Mimura and Asano, 1986 ; Imamura et al ., 1988 ; Baron et al ., 1987), an extended rodlike domain which is responsible for dimer formation and consists of four internal repeating units of 122 amino acids each (Mimura and Asano, 1987 ; Imamura. et al ., 1988), and a COOH-terminal domain containing two EFhand calcium-binding regions (Baron et al ., 1987; Noegel et al ., 1987) . Comparison ofa-actinin sequences with those of spectrins and dystrophin has revealed extensive homologies, leading to the suggestion that these three proteins are members of a single family of cytoskeletal proteins (Hammond, 1987 ; Koenig et al ., 1988 ; Davison and Critchley, 1988; Davison et al ., 1989) . In striated and smooth muscles, a-actinin is localized at Z-lines (Masaki et al ., 1967 ; Goll et al ., 1969) and dense bodies (Geiger et al ., 1981), respectively, where it seems to be involved in anchoring actin thin filaments. In cultured nonmuscle cells a-actinin is localized along actin stress fibers with a periodic distribution reminiscent of muscle sarcomeres (Lazarides and Burridge, 1975) . Here it may also be involved in anchoring actin filaments as well as bundling 481 on A ril 6, 2017 D ow nladed fom Published August 1, 1991