Bundling of actin filaments by α-actinin depends on its molecular length

Abstract

Cross-linking of actin filaments (F-actin) into bundles and networks was investigated with three different isoforms of the dumbbell-shaped α-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 α-actinin, but not Dictyostelium α-actinin under conditions where actin by itself polymerized into disperse filaments. This F-actin bundle formation critically depended on the proper molar ratio of α-actinin to actin, and hence F-actin bundles immediately disappeared when free α-actinin was withdrawn from the surrounding medium. The apparent dissociation constants (Kds) at half-saturation of the actin binding sites were 0.4 μM at 22°C and 1.2 μM at 37°C for chicken gizzard, and 2.7 μM at 22°C for both Acanthamoeba and Dictyostelium α-actinin. Chicken gizzard and Dictyostelium α-actinin predominantly cross-linked actin filaments in an antiparallel fashion, whereas Acanthamoeba α-actinin cross-linked actin filaments preferentially in a parallel fashion. The average molecular length of free α-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 α-actinin. In negatively stained preparations we also evaluated the average molecular length of α-actinin when bound to actin filaments: 36 nm for chicken gizzard and 35 nm for Acanthamoeba α-actinin, a molecular length roughly coinciding with the crossover repeat of the two-stranded F-actin helix (i.e, 36 nm), but only 28 nm for Dictyostelium α-actinin. Furthermore, the minimal spacing between cross-linking α-actinin molecules along actin filaments was close to 36 nm for both smooth muscle and Acanthamoeba α-actinin, but only 31 nm for Dictyostelium α-actinin. This observation suggests that the molecular length of the α-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 α-actinin molecules along the actin filaments.