Properties of the 120,000- and 95,000-dalton actin-binding proteins from Dictyostelium discoideum and their possible functions in assembling the cytoplasmic matrix.

Abstract

The cell cortex of Dictyostelium amebae contains an actin-rich cytoplasmic matrix. Changes in geometry of this matrix are believed to regulate protrusive activity and motility of the cell cortex . Two actin-binding proteins (120,000 and 95,000 daltons [120K and 950 are present in the cell cortex, and their properties, many of which are described here for the first time, suggest that they regulate growth and organization of cortical microfilaments . The 120K protein is a flexible dimer 35 nm in length with a native molecular mass of 241,000 . It nucleates the polymerization of actin and crosslinks the filaments to form branched networks like those seen in situ in the cell cortex . The production of a branched network of short crosslinked filaments results in a lattice that would theoretically generate the maximum rigidity with minimum amount of polymer . This sort of lattice would be very useful as a space-filling cytoskeleton capable of resisting deformation . The 120K protein inhibits the actin-stimulated Mg ATPase of myosin . Competition for actin binding between 120K and myosin, the impenetrability of the 120K-actin network to myosin, and the rigidity of actin filaments that are crosslinked by 120K could all contribute to the decrease in the actin-stimulated Mg ATPase of myosin . The properties of 120K are consistent with a role for this protein in regulating the site of actin filament growth and gelation in the cell but not the assembly of actin-containing structures that would participate in force generation by a sliding-filament mechanism involving myosin . The 95K protein is a rigid dimer 40 nm in length with a native molecular mass of between 190,000 and 210,000. Its physical and antigenic properties lead us to conclude that the 95K protein is Dictyostelium a-actinin . Unlike 120K, it crosslinks actin filaments into lateral arrays and increases the actin-stimulated Mg ATPase of myosin . Both activities are regulated by Cat+. The properties of 95K are consistent with a role in organizing actin filaments in the cell into lateral arrays that are capable of efficient interaction with myosin to produce force for cell motility .The cytoplasmic matrix is a pervasive filamentous network in the cytoplasm of many eukaryotic cells. This structure was first described by Wolosewick and Porter (42) as a microtrabecular lattice of heterogeneous morphology containing all three of the major filament classes (microfilaments, intermediate filaments, and microtubules) crosslinked to each other by narrow "trabeculae." Recently, workers in many labora-THE JOURNAL OF CELL BIOLOGY " VOLUME 99 NO . 1 PT . microfilaments and membranes, furtheremphasizing the view of the cytoplasm as a mechanically integrated network. These studies are largely summarized in the papers of this supplement.We have been studying the actin-rich zones of the cytomatrix in ameboid cells. In vegetative amebae of Dictyostelium discoideum this zone is found in the cell cortex . The cytoplasmic matrix in the cortex is heterogeneous, being composed o...

Novel Actin Cytoskeleton. Actin Tubules.

“…a-actinin and ABP-120 are F-actin cross-linking proteins in D. discoideum (Condeelis and Vahey, 1982;Condeelis et al, 1984). In mutants that lack either of them (Schleicher et al, 1988;Brink et al, 1990), spore actin rods were found both in the nucleus and the cytoplasm in a similar manner to that of the wild type strain (data not shown).…”

Re-expression of ABP-120 rescues cytoskeletal, motility, and phagocytosis defects of ABP-120- Dictyostelium mutants.

“…ABP and ABP-120 cross-link actin filaments in vitro to form a rigid actin gel containing orthogonal networks similar to those observed in pseudopods in situ (Condeelis et al, 1984;Hartwig and Shevlin, 1986;Wolosewick and Condeelis, 1986;Ogihara et al, 1988). Both proteins have also been localized to filament networks found in pseudopods in situ by immunoelectron microscopy (Hartwig and Shevlin, 1986; al., 1988;Cox et al, 1995).…”

“…The protein in Dictyostelium that is most likely to fulfill this cross-linking function is ABP-120. ABP-120 is a dimer consisting of two identical subunits each with a molecular weight of 120,000 on SDS-PAGE (Condeelis et al, 1984), which are packed in an anti-parallel orientation (Brink et al, 1990). Based on structural similarities, ABP-120 has been proposed to be a member of the filamin family of actin binding proteins, which is composed of ABP, ABP-280, and nonmuscle filamin (Hartwig and Kwiatkowski, 1991;Matsudaira, 1991).…”

Genetic deletion of ABP-120 alters the three-dimensional organization of actin filaments in Dictyostelium pseudopods.

“…ABP-120 is incorporated into the actin cytoskeleton at times after stimulation with cAMP that correlate with cross-linking of actin into the cytoskeleton and pseudopod extension Dharmawardhane et al, 1989). ABP-120 crosslinks actin filaments in vitro to form a rigid actin gel containing orthogonal networks similar to those observed in pseudopods in situ (Condeelis et al, 1984, Wolosewick andCondeelis, 1986;Ogihara et al, 1988).ABP-120 is a 40-nm rod-shaped protein consisting of two identical subunits each with a molecular mass of 120,000 D on SDS-PAGE (Condeelis et al, 1984) or 92,200 D as inferred from sequence analysis (Noegel et at., 1989). Each monomer contains an amino-terminal actin filament-binding site which is highly conserved throughout a number of actinbinding protein families (Bresnick et al, , 1991.…”

“…ABP-120 is a 40-nm rod-shaped protein consisting of two identical subunits each with a molecular mass of 120,000 D on SDS-PAGE (Condeelis et al, 1984) or 92,200 D as inferred from sequence analysis (Noegel et at., 1989). Each monomer contains an amino-terminal actin filament-binding site which is highly conserved throughout a number of actinbinding protein families (Bresnick et al, , 1991.…”