Abstract. Centractin (Arpl), an actin-related protein, is a component of the dynactin complex. To investigate potential functions of the protein, we used transient transfections to overexpress centractin in mammalian cells. We observed that the overexpressed polypeptide formed filamentous structures that were significantly longer and more variable in length than those observed in the native dynactin complex. The centractin filaments were distinct from conventional actin in subunit composition and pharmacology as demonstrated by the absence of immunoreactivity of these filaments with an actin-specific antibody, by resistance to treatment with the drug cytochalasin D, and by the inability to bind phalloidin. We examined the transfected cells for evidence of specific associations of the novel centractin filaments with cellular organelles or cytoskeletal proteins. Using immunocytochemistry we observed the colocalization of Golgi marker proteins with the centractin polymers. Additional immunocytochemical analysis using antibodies to non-erythroid spectrin (fodrin) and Golgi-spectrin ([3IE*) revealed that spectrin colocalized with the centractin filaments in transfected cells. Biochemical assays demonstrated that spectrin was present in dynactin-enriched cellular fractions, was coimmunoprecipitated from rat brain cytosol using antibodies to dynactin subunits, and was coeluted with dynactin using affinity chromatography. Immunoprecipitations and affinity chromatography also revealed that actin is not a bona fide component of dynactin. Our resuits indicate that spectrin is associated with the dynactin complex. We suggest a model in which dynactin associates with the Golgi through an interaction between the centractin filament of the dynactin complex and a spectrin-linked cytoskeletal network. CENTRACTIN is an actin-related protein (Arp) 1 that has been defined as a member of the Arpl class (Frankel et al., 1994;Fyrberg et al., 1994;Schroer et al., 1994). Actin-related proteins share 30-62% sequence identity with conventional actin and are predicted to have similar core structures for nucleotide binding (Kabsch and Holmes, 1995;Kelleher et al., 1995). The actin-related proteins have been grouped into three classes , however, the extent of the actinrelated protein family is not yet clear. To date, over 20 actin-related proteins have been identified in eukaryotes; an actin-related protein in protists has also been reported (Guerrero et al., 1995). The actin-related proteins appear to have important, but diverse cellular functions. Arpl null mutants in S. cerevisiae have defects in nuclear migration and mitotic spindle orientation

Cytoplasmic dynein is an intracellular motor responsible for endoplasmic reticulum-to-Golgi vesicle trafficking and retrograde axonal transport. The accessory protein dynactin has been proposed to mediate the association of dynein with vesicular cargo. Dynactin contains a 37-nm filament made up of the actin-related protein, Arp1, which may interact with a vesicle-associated spectrin network. Here, we demonstrate that Arp1 binds directly to the Golgi-associated ␤III spectrin isoform. We identify two Arp1-binding sites in ␤III spectrin, one of which overlaps with the actin-binding site conserved among spectrins. Although conventional actin binds weakly to ␤III spectrin, Arp1 binds robustly in the presence of excess F-actin. Dynein, dynactin, and ␤III spectrin co-purify on vesicles isolated from rat brain, and ␤III spectrin co-immunoprecipitates with dynactin from rat brain cytosol. In interphase cells, ␤III spectrin and dynactin both localize to cytoplasmic vesicles, co-localizing most significantly in the perinuclear region of the cell. In dividing cells, ␤III spectrin and dynactin co-localize to the developing cleavage furrow and mitotic spindle, a novel localization for ␤III spectrin. We hypothesize that the interaction between ␤III spectrin and Arp1 recruits dynein and dynactin to intracellular membranes and provides a direct link between the microtubule motor complex and its membrane-bounded cargo.The microtubule-based motor cytoplasmic dynein is involved in a wide range of cellular processes, including retrograde transport in neurons, trafficking of vesicles from the endoplasmic reticulum to the Golgi, mitotic spindle assembly, and potentially cytokinesis (reviewed in Ref. 1). These processes require the targeting of dynein to many different cellular cargoes. Whereas dynein clearly provides a motile force in all of these processes, the mechanisms linking dynein to these various cargoes have yet to be identified (reviewed in Ref. 2).Dynactin is a multi-subunit complex that binds both to microtubules (3) and to cytoplasmic dynein (4, 5). Disruption of the dynein-dynactin interaction blocks dynein-mediated transport both in vitro and in vivo (6 -9). The specific role of the interaction between dynein and dynactin is unknown. One possibility is that dynactin increases the processive nature of the movement of dynein along cellular microtubules (3, 10). A second but not mutually exclusive hypothesis is that dynactin links dynein to its cellular cargo (11).Although a dynactin-independent association between a dynein light chain and the rhodopsin receptor has been detected in rod cell vesicles (12), other studies suggest that dynactin is required to mediate the interaction of dynein with vesicular cargo. Antibodies that block the dynein-dynactin interaction deplete dynein from vesicles and inhibit microtubule-based vesicular transport (6, 7). Furthermore, disruption of the dynein-dynactin interaction by dynamitin overexpression inhibits dynein-mediated vesicle trafficking in cells (9). The nature of the interaction ...

The Arp2͞3 complex, a seven-subunit protein complex containing two actin-related proteins, Arp2 and Arp3, initiates formation of actin filament networks in response to intracellular signals. The molecular mechanism of filament nucleation, however, is not well understood. Arp2 and Arp3 are predicted to bind ATP via a highly conserved nucleotide-binding domain found in all members of the actin superfamily and to form a heterodimer than mimics a conventional actin dimer. We show here that adenosine nucleotides bind with micromolar affinity to both Arp2 and Arp3 and that hydrolyzable ATP is required for actin nucleation activity. Binding of N-WASP WA increases the affinity of both Arp2 and Arp3 for ATP but does not alter the stoichiometry of nucleotides bound in the presence of saturating concentrations of ATP. The Arp2͞3 complex bound to ADP or the nonhydrolyzable ATP analogue AMP-PNP cannot nucleate actin filaments, but addition of the phosphate analogue BeF 3 partially restores activity to the ADP-Arp2͞3 complex. Bound nucleotide also regulates the affinity of the Arp2͞3 complex for its upstream activators N-WASP and ActA. We propose that the active nucleating form of the Arp2͞3 complex is the ADP-P i intermediate in the ATPase cycle and that the ATPase activity of the Arp2͞3 complex controls both nucleation of new filaments and release of the Arp2͞3 complex from membraneassociated activators.T he actin cytoskeleton determines the shape, motility, and internal organization of eukaryotic cells. Many actin-based structures, especially those involved in membrane protrusion, are assembled by coordinated polymerization and crosslinking of new actin filaments from actin monomers into either orthogonal or parallel filament networks (1). In these structures, work is accomplished by the free energy of polymerization. Subsequent ATP hydrolysis by filamentous actin then allows the networks to be disassembled. The rapid and regulated assembly and disassembly of actin filament networks lies at the heart of many cellular processes that involve membrane protrusion such as cell locomotion, endocytosis, and phagocytosis (2).The Arp2͞3 complex plays a central role in the regulated assembly of actin-based structures. The Arp2͞3 complex nucleates formation of new actin filaments in response to upstream signaling events and simultaneously crosslinks them into orthogonal networks. Activation of Rho-family G proteins, including Rac and Cdc42, leads to dramatic reorganization of the actin cytoskeleton (3, 4). Rac and Cdc42 promote activation of members of the WASP family of proteins (5-7) that include WASP, N-WASP, and several isoforms of Scar (8). WASP family proteins, in turn, recruit and activate the Arp2͞3 complex (7, 9, 10). The Arp2͞3 complex nucleates formation of new actin filaments from the sides of older filaments, creating a dendritic network of crosslinked actin filaments in vitro (11,12) and in vivo (13).We proposed that activation of Arp2͞3 complex involves the two actin-related proteins, Arp2 and Arp3, forming a heterodim...

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