Bundling of Actin Filaments by ct-Actinin Depends on Its Molecular Length

Cross-linking of actin filaments (F-actin) into bundles and networks was investigated with three different isoforms of the dumbbell-shaped c~-actinin homodimer under identical reaction conditions. These were isolated from chicken gizzard smooth muscle, Acanthamoeba, and Dictyostelium, respectively. Examination in the electron microscope revealed that each isoform was able to cross-link F-actin into networks. In addition, F-actin bundles were obtained with chicken gizzard and Acanthamoeba a-actinin, but not Dictyostelium u-actinin under conditions where actin by itself polymerized into disperse filaments. This F-actin bundle formation critically depended on the proper molar ratio of a-actinin to actin, and hence F-actin bundles immediately disappeared when free ct-actinin was withdrawn from the surrounding medium. The apparent dissociation constants (Kos) at half-saturation of the actin binding sites were 0.4 t~M at 22°C and 1.2/zM at 37°C for chicken gizzard, and 2.7 ~M at 22°C for both Acanthamoeba and Dictyostelium c~-actinin. Chicken gizzard and Dictyostelium a-actinin predominantly cross-linked actin filaments in an antiparallel fashion, whereas Acanthamoeba t~-actinin cross-linked actin filaments preferentially in a parallel fashion. The average molecular length of free ot-actinin was 37 nm for glycerolsprayed/rotary metal-shadowed and 35 nm for negatively stained chicken gizzard; 46 and 44 nm, respectively, for Acanthamoeba; and 34 and 31 nm, respectively, for Dictyostelium c~-actinin. In negatively stained preparations we also evaluated the average molecular length of ct-actinin when bound to actin filaments: 36 nm for chicken gizzard and 35 nm for Acanthamoeba ct-actinin, a molecular length roughly coinciding with the crossover repeat of the twostranded F-actin helix (i.e, 36 nm), but only 28 nm for Dictyostelium o~-actinin. Furthermore, the minimal spacing between cross-linking c~-actinin molecules along actin filaments was close to 36 nm for both smooth muscle and Acanthamoeba o~-actinin, but only 31 nm for Dictyostelium ot-actinin. This observation suggests that the molecular length of the a-actinin homodimer may determine its spacing along the actin filament, and hence F-actin bundle formation may require "tight" (i.e., one molecule after the other) and "untwisted" (i.e., the long axis of the molecule being parallel to the actin filament axis) packing of a-actinin molecules along the actin filaments. I N 1964 ~-actinin was discovered as a protein extracted from striated muscle promoting contraction of actomyosin gels and increasing the viscosity of F-actin solutions in vitro (Ebashi et al., 1964). As more effective separation methods became available, its interaction with actin was more systematically investigated (Holmes et al., 1971; Goll et al., 1972). Accordingly, the largest increase in viscosity of a F-actin solution containing a given amount of ct-actinin was observed at 0°C. Under these conditions the viscosity reached a maximum at an ot-actinin to actin ratio yielding about one a-actinin dimer molecule bound per crossover repeat (i.e., 36 nm) of the actin helix. A much higher o~-actinin to actin ratio was needed to yield the same amount of bound o~-actinin in solutions kept at 37°C. ot-Actinin is a homodimer composed of two polypeptides of ~100 kD each (Suzuki et al., 1976). Electron micrographs of shadowed ot-actinin have revealed a dumbbellshaped molecule with the two subunits being oriented antiparallel in a side-by-side association thus having a central dyad axis of symmetry (e.g., Pollard et al., 1986). Each polypeptide has a highly conserved actin binding site located near the NH2-terminai of the polypeptide chain that on the molecule is located on the "knob-like" protrusion at the end of the rod (Mimura and Asano, 1987; Imamura et al., 1988; Blanchard et al., 1989). As a consequence, o~-actinin crosslinks F-actin by binding with each end to an actin filament (podlubnaya et al., 1975). Over the past few years many more a-actin isoforms have been isolated and characterized (Feramisco and Burridge, 1980; Burridge and Feramisco, 1981; Pollard, 1981; Condeelis and Vahey, 1982; Duhaiman and Bamburg, 1984; Schleicher et al., 1984), and broadly cross-reacting antibodies have been raised (Lazarides and Burridge, 1975). The o~-actinin-actin interaction of most nonmuscle isoforms was © The Rockefeller University Press, 0021-9525/90/06/2013/12 $2.00 The Journal of Cell Biology, Volume 110, June 199