Three unrelated tumor cell lines derived from human malignant melanomas lack actin-binding protein (ABP), which cross-links actin filaments in vitro and connects these filaments to plasma membrane glycoproteins. The ABP-deficient cells have impaired locomotion and display circumferential blebbing of the plasma membrane. Expression of ABP in one of the lines after transfection restored translocational motility and reduced membrane blebbing. These findings establish that ABP functions to stabilize cortical actin in vivo and is required for efficient cell locomotion.

Abstract. nonmuscle filamin) is a ubiquitous dimeric actin cross-linking phosphoprotein of peripheral cytoplasm, where it promotes orthogonal branching of actin filaments and links actin filaments to membrane glycoproteins. The complete nucleotide sequence of human endothelial cell ABP cDNA predicts a polypeptide subunit chain of 2,647 amino acids, corresponding to 280 kD, also the mass derived from physical measurements of the native protein. The actin-binding domain is near the amino-terminus of the subunit where the amino acid sequence is similar to other actin filament binding proteins, including t~-actinin,/3-spectrin, dystrophin, and Dictyostelium abp-120. The remaining 90% of the sequence comprises 24 repeats, each ~96 residues long, predicted to have stretches of/3-sheet secondary structure interspersed with turns. The first 15 repeats may have substantial intrachain hydrophobic interactions and overlap in a staggered fashion to yield a backbone with mechanical resilience. Sequence insertions immediately before repeats 16 and 24 predict two hinges in the molecule near points where rotaryshadowed molecules appear to swivel in electron micrographs. Both putative hinge regions are susceptible to cleavage by proteases and the second also contains the site that binds the platelet glycoprotein Ib/IX complex. Phosphorylation consensus sequences are also located in the hinges or near them. Degeneracy within every even-numbered repeat between 16 and 24 and the insertion before repeat 24 may convert interactions within chains to interactions between chains to account for dimer formation within a domain of 7 kD at the carboxy-terminus. The structure of ABP dimers resembles a leaf spring. Interchain interactions hold the leaves firmly together at one end, whereas intrachain hydrophobic bonds reinforce the arms of the spring where the leaves diverge, making it sufficiently stiff to promote high-angle branching of actin illaments. The large size of the leaves, their interruption by two hinges and flexible actin-binding site, facilitate cross-linking of widely dispersed actin filaments.N ONMUSCLE cells achieve a wide variety of shapes by arranging peripheral linear actin filaments in different ways. Actin-modulating proteins are responsible for this versatility by adjusting actin filament length and by defining the organization of actin filaments and their binding to other cellular components (54, 56). Actin-binding protein (ABE also called nonmuscle filamin) ~ was among the first such proteins discovered (25,55,62) and has a broad phylogenetic distribution (3,4,34,47,49,51,59,60,63). ABP from most cell types examined promotes high-angle

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