A 13-/ Map of the Actin-Scruin Filament from the Limulus Acrosomal Process

We have determined the structure of the actin-scruin filament to 13-/k resolution using a combination of low-dose EM and image analysis. The threedimensional map reveals four actin-actin contacts: two within each strand and two between strands. The conformation of the actin subunit is different from that in the Holmes et al. (1990) model as refined by Lorenz et al. (1993). In particular, subdomain II is tilted in a similar way to that seen by Orlova and Egelman (1993) in F-Mg2÷-ADP actin filaments in the absence of Ca 2÷. Scruin appears to consist of two domains of approximately equal volume. Each scruin subunit cross-links the two strands in the actin filament. Domain I of scruin contacts subdomain I of actin and makes a second contact at the junction of subdomains In and IV. Domain II of scruin contacts actin at subdomains I and II of a neighboring actin subunit. The two scruin domains thus bind differently to actin. TIS is a structural protein involved in cell motility and shape. The utility of actin lies in its abili~ to bind different actin binding proteins and generate different functional assemblies. Examples include the contractile apparatus of muscle (Huxley, 1969), microvillar bundles of intestinal epithelial cells (Maroux et al., 1988; Burgess, 1987), stereocilia of the inner ear (Flock and Cheung, 1977; DeRosier et al., 1980), the acrosomal process of Limulus sperm (Tilney, 1975), bundles within the filopodia of Dictyostelium (Demma et al., 1990; Dharmawardhane et al., 1991; Owen et al., 1992), and the erythrocyte cytoskeleton (Branton et al., 1981). Recent findings suggest that actin structures can also provide indirect control of cellular processes. For instance, EF-la, which is an important component of protein synthesis, is also an actin bundling protein that is associated with locomotion in Dictyostelium (Yang et al., 1990; Dharmawardhane et al., 1991). Knowledge of the high resolution structure is necessary for understanding how actin can be involved in different structures and interactions. Much has been learned over the past 15 yr by EM and image analysis (Moore et al., 1970; Vibert and Craig, 1982; Bullitt et al., 1988; Milligan et al., 1990), but the recent atomic structure of an actin:DNase I co-crystal (Kabsch et al., 1990) is facilitating the interpretation of much of the existing data (Holmes et al., 1990; Lorenz et al., 1993; Rayment et al., 1993; Schroder et al., 1993). It allows us to consider how the subdomains of actin interact with the various actin binding proteins. Are the subdomains locked or free to move, as suggested by normal mode analysis of G-actin (Tirion and ben-Avraham, 1993)? How does the tertiary conformation change with different actin binding proteins? EM is ideally suited for looking at actin in different assemblies. Actin filaments are helical structures, and by the nature of a helix provide three-dimensional information from a single view (DeRosier and Moore, 1970). A resolution of 25/~ radially by 45/~ axially has been sufficient to make detailed observations of the actin filament bound with myosin subfragment-1 (S1) and tropomyosin (Milligan et al., 1987, 1990); thin filaments plus invertebrate myosin S1 with and without regulatory light chain (Vibert and Craig, 1982); and F-actin in which evidence has been found for a conformational change dependent on the nucleotide (Orlova and Egelman, 1992) or the divalent cation (Mg 2÷ vs Ca 2+) (Orlova and Egelman, 1993). We have studied F-actin filaments bound with the ll0-kD Limulus acrosomal process protein scruin (Tilney, 1975; Bullitt et al., 1988). The acrosomal process is a 60-#m-long actin bundle composed of ~o100 hexagonally packed actin filaments, wound into a supercoil about the base of the nucleus. Upon stimulation, the supercoil is released, and the bundle rapidly uncoils and extends through the nucleus and out the anterior end of the sperm to penetrate the egg. Recently, the gene for scruin has been cloned and sequenced (M. Way and P. Matsudaira, personal communication). They found it is a 103-kD protein containing two domains that have arisen through gene duplication. Scruin is related to the protein kelch which has one rather than two domains. Kelch is found in the ring canal between the nurse cell and the developing oocyte in Drosophila (Xue and Cooley, 1993). Our goal in this work was to extend the resolution of previous work to better resolve the actin structure and conformation and better identify its interaction with scruin. We carried this out by collecting Fourier data for layer lines beyond the resolution at which reflections were obvious above the © The Rockefeller University Press, 0021-9525/93/10/337/8 $2.00 The Journal of Cell Biology, Volume 123, Number 2, October 1993 337-344 337 background. We then averaged many such data sets to improve the signal/noise ratio and calculated a 13-,~ map. We fit the atomic model for actin to our map, in order to (a) define the boundary between actin and scruin, (b) determine the contact sites between actin and scruin subunits, and (c) make a detailed comparison of the actin monomer in this actin/scruin structure with that in other F-actin containing structures and with the monomer in an actin/DNase I structure. Materials and Methods Preparation of Acrosomal Processes Sperm from Limulus polyphemus were collected as described previously (Tilney, 1975). Sperm were washed in collecting media (423 mM NaC1, 10 mM KCI, 2 mM MgC12, 5 mM NaPO4, pH 8.0), then resuspended in TPM (1% Triton X-100 [Sigma Immunochemicals, St. Louis, MO], 5 mM NaPO4, 2 mM MgC12, 0.1 mM EDTA, 0.2 mM ATE pH 8.0), which induces the false discharge. Acrosomal processes were sheared off by sucking repeatedly up and down a Pasteur pipette. Sperm nuclei and attached flagellar axonemes and acrosomal caps were removed by centrifugation for 5 min at 750 g. The supernate contains primarily acrosomal processes, with an occasional flagellar axoneme or nucleus, as judged by light microscopy. Acrosomal processes were centrifuged for 10 min at 10,000 g and the pellet resuspended in F-actin buffer (2 mM MgC12, 10 mM KCI, 5 mM NaPO4, 0.2 mM ATE pH 7.5). The acrosomal processes were then centrifuged and resuspended in a small quantity of G-actin buffer (2 mM Tris, 0.2 mM CaCl2, 0.5 ram DTT, 0.2 mM ATE 1 /zg/ml leupeptin, pH 8.2) and dialyzed for several hours against G-actin buffer that included 0.1 mM PMSE G-actin buffer caused bundles to dissociate into single actin-scrniu filaments, as judged by EM. SDS Gel Electrophoresis Undissociated bundles were removed from the dissociated bundle suspension by centrifuging for 3 min in a desktop microfuge. Filaments were analyzed by discontinuous SDS-PAGE with a 5% stacking gel and a 12% resolving gel (Laemmli, 1970).