Gene name - rhea
Synonyms - Talin
Cytological map position - 66D6
Function - cytoskeletal anchor protein
Symbol - rhea
FlyBase ID: FBgn0260442
Genetic map position -
Classification - FERM domain, PTP domain, I/LWEQ module (actin-interacting domain)
Cellular location - cytoplasmic
The gene rhea, isolated because of its wing blister phenotype (Prout, 1997), is typical of mutants affecting integrin function, and it encodes Drosophila Talin (Brown, 2002). Talin was identified in a differential embryonic head cDNA screen (Brody, 2000 and 2002) and is expressed in a subset of CNS neurons. Embryos deficient in Talin have very similar phenotypes to integrin (ßPS) null embryos, including failure in germ band retraction and muscle detachment, however, no CNS-specific phenotype is detected (Brown, 2002). Talin is not required for the presence of integrins on the cell surface or their localization at muscle termini. However, Talin is required for formation of focal adhesion-like clusters of integrins on the basal surface of imaginal disc epithelia and junctional plaques between muscle and tendon cells. These results indicate that Talin is essential for integrin function and acts by stably linking clusters of ECM-linked integrins to the cytoskeleton (Brown, 2002).
Integrin-mediated adhesion between the cellular cytoskeleton and the extracellular matrix (ECM) is vital to tissue interactions. Integrin adhesion mediates stable attachment between tissue layers, as shown by defects caused by integrin gene mutations (Brown, 2000; Hogg, 2000; Sheppard, 2000). In flies and mice, a key role for integrins in tissue adhesion is to attach the ends of striated muscles to the ECM, as shown by muscle detachment in the absence of integrins. Loss of integrins also leads to separation between epidermis and dermis, causing epidermolysis bullosa in humans. In the fly, loss of integrins causes separation of the two epithelial layers that form the wing. Integrin-mediated adhesion involves three elements: ECM, integrins, and cytoskeleton. Defects in any of these elements lead to failure of adhesion and separation of tissue layers (Brown, 2002 and references therein).
Given the strong tensile stresses placed on the integrin linkage, the short cytoplasmic tails of integrins seem inadequate to connect to the cytoskeleton. The ß subunit of the integrin heterodimer makes this connection, in most cases, with a cytoplasmic domain of <50 amino acids. Despite the small size of the integrin cytoplasmic domain, numerous proteins bind directly to this domain. A key binding protein, talin, was the first to be identified (Horwitz, 1986). Talin is enriched in focal adhesions: integrin-adhesive structures found in cells spread on ECM. Talin is a large polypeptide of greater than 2500 amino acids, composed of an ~50 kDa N-terminal head and an ~220 kDa C-terminal rod, and has several functional domains (Rees, 1990), two of which are shared with other proteins that link membranes to the cytoskeleton. The head has a FERM domain, shared with proteins such as moesin and band4.1 (Chishti, 1998), which has binding sites for proteins and phosphoinositides (Pearson, 2000). Most of the rod sequence consists of alanine-rich repeats (McLachlan, 1994). At the C terminus there is an I/LWEQ domain (McCann, 1997) shared between talin and a protein involved in endocytosis: the yeast actin binding protein Sla2 and its vertebrate homologs, Huntingtin-interacting protein 1 (Hip1) and Hip1-related protein (Brown, 2002 and references therein).
Talin has sites in the head and rod that bind to integrin cytoplasmic tails (Horwitz, 1986; Calderwood, 1999; Patil, 1999; Yan, 2001). Talin recruitment to clusters of integrins requires ligand occupancy (Miyamoto, 1995). In C. elegans, talin recruitment to sites of integrin adhesion requires integrins (Moulder, 1996). Talin has binding sites for a number of other proteins. Focal adhesion kinase, vinculin, and actin bind to talin in vitro, with several sites identified for these proteins (Hemmings, 1996; Borowsky, 1998; Bass, 1999). When calpain cleaves talin into head and rod fragments, only the rod interacts strongly with actin (Niggli, 1994). When expressed in cells, the N-terminal actin binding region associates weakly with stress fibers, while the C-terminal domain localizes to focal adhesions (Hemmings, 1996). In vitro talin has diverse effects on actin, including nucleating, shortening, and crosslinking actin filaments (Goldmann, 1994, 1999). The transmembrane lectin layalin also interacts with the head, showing that talin interaction with membrane proteins is not limited to integrins (Borowsky, 1998). Talin can also interact directly with lipid membranes (Niggli, 1994). The numerous binding sites of talin make it difficult to pose models for the topology of talin in focal adhesions, particularly because it forms antiparallel homodimers (Isenberg, 1998). Cleaving talin into head and tail fragments increases binding to the integrin ß subunit cytoplasmic tail, suggesting that, in the intact molecule, the tail folds back and inhibits the head, as has been seen in other FERM domain proteins (Brown, 2002 and references therein).
Talin function has been studied by antibody inhibition and genetic analysis. Anti-talin antibodies prevents formation of focal adhesions (Nuckolls, 1992), and antisense inhibition reduces focal adhesion size and the rate of integrin processing and transport to the cell surface (Albiges-Rizo, 1995; Martel, 2000). The slime mold Dictyostelium has two talin genes with different functions. Talin A mutants developed normally (Niewohner, 1997); talin B mutants do not develop beyond the mound stage (Tsujioka, 1999). Talin A mutants fail to adhere to nonspecific substrates and to phagocytose particles (Niewohner, 1997). Thus, talin A has a conserved function in substrate adhesion, although this may be mediated by receptors other than integrins. Knockout of talin in the mouse resulted in only a partial knockout of function, most likely due to a second, recently identified talin gene (Monkley, 2001). Undifferentiated talin1 knockout cells did not form focal adhesions but, after differentiation, can form them (Priddle, 1998). Mouse embryos lacking talin1 appear disorganized after gastrulation and are then resorbed, but this phenotype is milder than that of embryos lacking ß1 integrin (Monkley, 2000). Cells derived from these talin1 homozygous mutant embryos have reduced focal adhesions, which still stain for talin. These results emphasize that a complete knockout of talin will be essential to fully understand its role in focal adhesion formation. Genetics has so far supported a key role for talin, but in neither Dictyostelium nor the mouse have the consequences of a complete absence of talin been shown. From the genome sequence of Drosophila, it is known that there is a single talin gene, making the fly an excellent system for analysis of talin function (Brown, 2002).
A genetic strategy was used to identify proteins required for integrin-mediated adhesion. Genetic screens were performed for mutations that, like integrins, cause a failure in adhesion between the two layers of the wing. Homozygous lethal mutants were recovered by screening mosaic flies carrying clones of homozygous mutant cells in the wing. Mutations in 32 different loci were isolated (23 with multiple alleles) including the three integrin loci. One novel locus was named rhea, after the flightless bird. The rhea locus has been shown to correspond to the single Drosophila talin gene. As observed in other systems, talin colocalizes with integrins in an integrin-dependent manner. Generally, loss of talin closely mimics the loss of integrins, indicating that talin is an obligatory component of integrin-mediated adhesion (Brown, 2002).
These analyses contribute three main findings. (1) Talin is an essential core component of the integrin-mediated adhesion mechanism: equivalent phenotypes are caused by absence of talin or the ßPS integrin subunit. (2) The key function of talin is to connect ECM-bound integrins to the actin cytoskeleton, and it is not required for the preceding localization of integrins to the cell surface or binding to the ECM. (3) Integrin signaling to modulate gene expression required little, if any, talin function, suggesting that integrin connection to the cytoskeleton is not obligatory for signaling (Brown, 2002).
The rhea gene is the third 'wing blister' gene found to encode an adaptor protein that mediates attachment of ECM-integrin complexes to the actin cytoskeleton. The other two are short stop (shot, previously known as kakapo) and integrin linked kinase (ilk). The shot gene encodes a protein related to spectrin, dystrophin, bullous pemphagoid antigen 1, and plectin that has both actin and microtubule binding regions (Fuchs, 2001). ILK has an ankryin repeat domain and a kinase-like domain, and, based on its subcellular localization, phenotype, and binding partners, it also plays a role in the link between integrins and the cytoskeleton. However, the embryonic phenotypes of mutations in these three genes differ significantly. While talin and integrin mutants have essentially indistinguishable phenotypes, ilk and shot mutants have only a subset of these defects. Embryos lacking ILK only have a muscle detachment phenotype, which is milder and appeared later in development, as the force of muscle contraction has increased, suggesting that integrin-mediated adhesion is weakened, rather than eliminated. Shot is essential for the attachment of the tendon cell transcellular microtubules to the basal integrin junctions but has no role in muscle cells, showing that it contributes to integrin adhesion in some, but not all, cells. Shot is also involved in integrin-independent processes, especially in the nervous system. These phenotypic differences between rhea, shot, and ilk emphasize the essential role of talin. In addition, talin is the first protein that has been found to require integrin function for its localization at muscle attachment sites. Thus, it is envisioned that integrins and talin form an interdependent core complex, which uses ILK and Shot as accessory proteins to make diverse connections with the cytoskeleton (Brown, 2002).
While talin was the first intracellular integrin binding protein to be identified, its significance was eclipsed by the identification of many other proteins that share this feature (Liu, 2000). At least two other proteins share with talin the ability to bind directly to integrin cytoplasmic domains and actin: alpha-actinin and filamin. This work reestablishes talin's central role in the integrin-cytoskeleton link. The potential functions of talin in integrin-mediated processes have expanded beyond linking integrins with the actin cytoskeleton, to include transport of integrins to the cell surface and activation of integrin binding to the ECM (inside-out activation). The results of this study suggest these other roles may not be as universal (Brown, 2002).
Talin is not required for assembly and transport of integrin heterodimers to the cell surface in the embryonic muscles. A defect in this process is easily observed in the muscles: in the absence of the only alpha subunit expressed in the muscles, alphaPS2, the ßPS subunit is observed in a perinuclear pattern, consistent with retention in the endoplasmic reticulum. This contrasts with the data for talin; in the absence of talin, PS2 integrin localizes to the muscle ends. Thus, a role for talin in integrin synthesis appears to be specific to certain kinds of cells (Brown, 2002).
It is also inferred from these results that talin is not essential for the adhesion of integrins to the ECM. Overexpression of the talin head stimulates inside-out signaling (Calderwood, 1999), suggesting that talin might be required for inside-out activation of integrins. However, absence of talin caused a break in the integrin-mediated adhesion mechanism between the integrin cytoplasmic tails and the actin cytoskeleton, not between integrins and the ECM. This suggests that either talin is not required for inside-out activation, because another protein substitutes for this function, or inside-out activation is not required for PS2 integrin adhesion to the tendon matrix. While constitutive activation of the PS2 integrin causes too much muscle attachment, it is not known whether a lack of inside-out activation causes a defect in muscle adhesion (Brown, 2002).
This study identified a role for talin in the aggregation of integrins into focal adhesion-like structures on the basal surface of the wing disc epithelia. It is envisioned that ligand-bound integrins contract into the focal adhesion-like structures by talin, either directly or through an interaction with the cytoskeleton. It is possible that talin is performing a similar role at the ends of muscles: it was not possible to observe the loss of aggregation because of talin-independent concentration of integrins to muscle termini. This is the first time focal adhesion structures containing integrins have been reported in the fly, but their function is not clear. Clones of cells lacking integrins or talin do not perturb the morphology of the disc or the proliferation of the cells in any obvious way. Analysis of integrin mutant clones has shown that the first phenotype observed during wing development is during the reapposition of the two surfaces of the wing during pupal development, when the mutant cells fail to reattach. An identical phenotype is seen in talin mutant clones (Brown, 2002).
These results suggest that talin is not required for integrin-mediated signaling to regulate gene expression. The assay available in Drosophila requires development of different regions of the midgut, which fails in the complete absence of talin. Removal of the zygotic talin causes gut defects similar to those caused by integrin alpha subunit mutations, indicating that residual maternal talin is not sufficient to mediate integrin adhesion. Despite this, integrin signaling is not impaired, suggesting that signaling downstream occurs parallel to talin and that the clustering of integrins associated with signaling is not mediated by talin. Before it can be said with complete confidence that talin has no role in signaling, an assay is required suitable for embryos that completely lack talin (Brown, 2002).
In summary, the data demonstrate that talin has a specific role in linking integrins to the cytoskeleton. Two models for talin function are consistent with the data presented: (1) talin aggregates integrins into clusters that recruit other proteins required for the cytoskeletal link, and (2) talin directly links integrins to actin (Brown, 2002).
This work provides further confirmation that basic mechanisms of integrin function are well conserved between invertebrates and vertebrates (Brown, 2000). In Drosophila, the structure of talin is highly similar to the vertebrate protein, containing integrin, vinculin, and actin binding domains, indicating the profound evolutionary maintenance of integrin-talin-dependent adhesive functions. Evidence is availible, mostly from in vivo studies, of the conserved function of these domains. For example, the binding of integins to talin is implied by the integrin-dependent recruitment of talin to the cell surface. In the absence of talin in muscles, actin is no longer colocalized with integrins, consistent with the view that talin links actin to integrins. Finally, it has been shown in blotting experiments that mammalian vinculin binds to Drosophila talin. The conservation of mechanism even extends into the synthesis of the components. In wings, expression of the Drosophila serum response factor (DSRF) is required for the formation of integrin-mediated attachment between the wing surfaces. In mouse, SRF is essential for talin and zyxin expression and is required for normal cell adhesion. Thus, further work on the role of talin in Drosophila should provide general insight into the role of this important cytoskeletal molecule (Brown, 2002).
Drosophila talin was first identified from Berkeley Drosophila Genome Project cDNA sequences. Subsequently, a set of exons were identified in the genome sequence that encodes a protein homologous throughout its length with talin from other species (CG6831). The transcript polyadenylation site was found from three independent ESTs, but the 5' end of the transcription unit has not been determined. A putative splice acceptor is found upstream of the initiating ATG, suggesting the existence of one or more 5' noncoding exons in the large area separating the talin coding region from the next predicted gene upstream (Brown, 2002).
An alignment of talin sequences from mouse, chicken, Drosophila, and C. elegans shows strong conservation in three areas. The N-terminal head has a large block that includes the FERM domain, which has binding sites for integrins, actin, and layalin. Within the rod is a second well-conserved domain, containing one of the vinculin binding sites. The third conserved domain overlaps the I/LWEQ actin binding site. The Drosophila protein is longer than the other talins in the alignment (there are an additional 284 residues). The extra sequence is not similar to any other protein (Brown, 2002).
date revised: 20 March 2003
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