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Images (GJ) depict a translationalfln-2c::gfpfusion. an N-terminal actin-binding domain name (ABD) followed by multiple immunoglobulin-like repeats (IgFLN). P505-15 (PRT062607, BIIB057) The best-characterized filamins areDictyostelium discoideumfilamin (ddFLN) and human filamins (hsFLNA/B/C).Dictyosteliumfilamin has an ABD followed by six IgFLN repeats, whereas the human orthologs have 24 IgFLN repeats arranged into two rod domains separated by a flexible hinge. FLNA, FLNB, and FLNC are more than 70% identical at the amino acid sequence level and have overlapping expression patterns. Although FLNA and FLNB are ubiquitously expressed, FLNC is found primarily in cardiac and striated muscle mass[1]. Filamins are involved in diverse cellular processes including anchoring, organizing and maintaining the actin cytoskeleton, providing a scaffold for signaling components, and acting as molecular sensors for mechanical causes[1]. Due to the pleiotropic functions of filamins in humans, mutations cause a wide variety of developmental defects in the skeleton, brain, heart, and easy muscle mass[2]. Although no total structure of a filamin molecule is available, biochemical and structural studies have provided important insights into the function of filamins[3],[4],[5]. The best-studied role of filamin is in the organization of actin filaments into branched three-dimensional networks[1]. Filamin binds F-actin using the N-terminal ABD, although some IgFLN repeats and hinge regions may also contribute to actin binding[6]. The filamin ABD consists of two calponin homology (CH) domains that are well conserved among filamins and other actin binding proteins, such as alpha-actinin, spectrin, and fimbrin[7]. In filamin, the primary actin-binding site is usually hydrophobic and is located in the first CH domain name (CH1)[8],[9],[10]. The second CH domain (CH2) has a lower affinity for actin, but is required for a fully functional ABD[10],[11]. Although CH2 is usually less conserved across filamins than CH1, disease-related mutations suggest that CH2 may regulate the actin-binding activity of CH1[12]. For example, gain-of-function mutations in the CH2 domain name of FLNA lead to developmental disorders of the skeleton by increasing P505-15 (PRT062607, BIIB057) filamin affinity for F-actin, which perturbs actin cytoskeleton dynamics[13]. Individual IgFLN repeats are 96 amino acids in length and are comprised of seven -strands (AG) arranged into two -linens, which together form a -sandwich. Filamins are predicted to interact with more than fifty different proteins, many of which interact with the CD strands of the IgFLN domains[14]. The majority of these interactions involve IgFLN domains in the second rod domain (IgFLN1624). For example, filamin binds transmembrane proteins such as integrins[15], transmembrane receptors[16], and many signaling proteins, including the Rho-family of GTPases[17],[18]. The cytoplasmic tail of 7 integrin binds to the CD face of FLNA IgFLN21[5], which links the actin network actually with the extracellular matrix (ECM). FLNA IgFLN24 binds RhoA, Rac1 and Cdc42, all of which regulate actin dynamics. In addition, the final repeat also mediates dimerization of filamins[6],[19],[20]. FLNB has also been shown to serve as a scaffold for signaling pathway components, for example, the Rac1, MEKK1, MKK4, and JNK cascade in interferon-induced apoptosis[1],[21],[22]. We are using the nematodeC. elegansas a model system to study the conserved functions of filaminin vivo.C. elegansprovides many advantages for the study of cytoskeletal regulation, including a translucent body, good visualization tools, and freely available genomic resources[23]. TheC. elegansgenome encodes two filamin homologs,fln-1andfln-2. In addition to filamins, many other cytoskeletal regulators are well conserved, including integrin[24], talin[25], vinculin[26], and the Rac GTPases[27]. We have recently shown that FLN-1 is required for maintenance of actin and for proper function P505-15 (PRT062607, BIIB057) of the spermatheca, a somatic tissue of the nematode reproductive system that undergoes dramatic shape changes during the ovulation and fertilization of oocytes[28]. The secondC. elegansfilamin ortholog, FLN-2, has not been studied, but large-scale RNAi studies suggest it may play a role in molting[29],[30]. In this study, we characterize the gene structures and expression patterns offln-1andfln-2,use homology modeling to determine the structure of the conserved IgFLN domains in these molecules, predict the conservation of function betweenC. elegansand other filamins, and demonstrate that this ABD of FLN-1 and FLN-2 can co-localize with actinin vivo. == Results == == TheC. elegansgenome encodes two filamin genes,fln-1andfln-2 == In order to identify filamin-like genes inC. elegans, we performed a BLAST CD63 search of theC. elegansgenome (WormBase release WS190) using human FLNA. At the time, this search recognized a cluster of predicted open reading frames (ORF) on chromosome IV (Y66H1B.2, Y66H1B.5, and Y66H1B.3;Physique 1A) and another on chromosome X (C23F12.1 and C23F12.2;Physique 1B). These ORFs lack one or more of the characteristic features of filamin,.