rhea/Talin: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - rhea

Synonyms - Talin

Cytological map position - 66D6

Function - cytoskeletal anchor protein

Keywords - integrin signaling, junctions, focal adhesion, cytoskeleton

Symbol - rhea

FlyBase ID: FBgn0260442

Genetic map position -

Classification - FERM domain, PTP domain, I/LWEQ module (actin-interacting domain)

Cellular location - cytoplasmic

NCBI link: Entrez Gene
rhea orthologs: Biolitmine
Recent literature
Vanderploeg, J. and Jacobs, R. (2015). Talin is required to position and expand the luminal domain of the Drosophila heart tube. Dev Biol [Epub ahead of print]. PubMed ID: 25958089
Fluid- and gas-transporting tubular organs are critical to metazoan development and homeostasis. Tubulogenesis involves cell polarization and morphogenesis to specify the luminal, adhesive, and basal cell domains and to establish an open lumen. This study explores a requirement for Talin, a cytoplasmic integrin adaptor, during Drosophila melanogaster embryonic heart tube development. Talin marked the presumptive luminal domain and was required to orient and develop an open luminal space within the heart. Genetic analysis demonstrated that loss of zygotic or maternal-and-zygotic Talin disrupted heart cell migratory dynamics, morphogenesis, and polarity. Talin was essential for subsequent polarization of luminal determinants Slit, Robo, and Dystroglycan as well as stabilization of extracellular and intracellular integrin adhesion factors. In the absence of Talin function, mini-lumens enriched in luminal factors formed in ectopic locations. Rescue experiments performed with mutant Talin transgenes suggested actin-binding was required for normal lumen formation, but not for initial heart cell polarization. The study proposes that Talin provides instructive cues to position the luminal domain and coordinate the actin cytoskeleton during Drosophila heart lumen development.
Hakonardottir, G. K., Lopez-Ceballos, P., Herrera-Reyes, A. D., Das, R., Coombs, D. and Tanentzapf, G. (2015). In vivo quantitative analysis of Talin turnover in response to force. Mol Biol Cell 26: 4149-4162. PubMed ID: 26446844
Cell adhesion to the extracellular matrix (ECM) allows cells to form and maintain three-dimensional tissue architecture. Cell-ECM adhesions are stabilized upon exposure to mechanical force. This study used quantitative imaging and mathematical modeling to gain mechanistic insight into how integrin-based adhesions respond to increased and decreased mechanical forces. A critical means of regulating integrin-based adhesion is provided by modulating the turnover of integrin and its adhesion complex (integrin adhesion complex [IAC]). The turnover of the IAC component Talin, a known mechanosensor, was analyzed using fluorescence recovery after photobleaching. Experiments were carried out in live, intact flies in genetic backgrounds that increased or decreased the force applied on sites of adhesion. This analysis showed that when force is elevated, the rate of assembly of new adhesions increases such that cell-ECM adhesion is stabilized. Moreover, under conditions of decreased force, the overall rate of turnover, but not the proportion of adhesion complex components undergoing turnover, increases. Using point mutations, the key functional domains of Talin were identified that mediate its response to force. Finally, by fitting a mathematical model to the data, the mechanisms that mediate the stabilization of ECM-based adhesion during development were uncovered.
Park, S. H., Lee, C. W., Lee, J. H., Park, J. Y., Roshandell, M., Brennan, C. A. and Choe, K. M. (2018). Requirement for and polarized localization of integrin proteins during Drosophila wound closure. Mol Biol Cell: mbcE17110635. PubMed ID: 29995573
Wound re-epithelialization is an evolutionarily conserved process in which skin cells migrate as sheets to heal the breach, and is critical to prevent infection, but impaired in chronic wounds. Integrin heterodimers mediate attachment between epithelia and underlying extracellular matrix, and also act in large signaling complexes. The complexity of the mammalian wound environment and evident redundancy among integrins has impeded determination of their specific contributions to re-epithelialization. Taking advantage of the genetic tools and smaller number of integrins in Drosophila, a systematic in vivo analysis of integrin requirements in the re-epithelialization of skin wounds was underrtaken in the larva. αPS2-βPS and αPS3-βPS were identified as the crucial integrin dimers, and talin was identified as the only integrin adhesion component required for re-epithelialization. The integrins rapidly accumulate in a JNK-dependent manner in a few rows of cells surrounding a wound. Intriguingly, the integrins localize to the distal margin in these cells, instead of the frontal or lamellipodial distribution expected for proteins providing traction, and also recruit nonmuscle myosin II to the same location. These findings indicate that signaling roles of integrins may be important for epithelial polarization around wounds, and lay the groundwork for using Drosophila to better understand integrin contributions to re-epithelialization.
Ren, Q. and Rao, Y. (2022). The exit of axons and glial membrane from the developing Drosophila retina requires integrins. Mol Brain 15(1): 2. PubMed ID: 34980203
Coordinated development of neurons and glia is essential for the establishment of neuronal circuits during embryonic development. In the developing Drosophila visual system, photoreceptor (R cell) axons and wrapping glial (WG) membrane extend from the eye disc through the optic stalk into the optic lobe. Extensive studies have identified a number of genes that control the establishment of R-cell axonal projection pattern in the optic lobe. The molecular mechanisms directing the exit of R-cell axons and WG membrane from the eye disc, however, remain unknown. This study shows that integrins are required in R cells for the extension of R-cell axons and WG membrane from the eye disc into the optic stalk. Knockdown of integrins in R cells but not WG caused the stalling of both R-cell axons and WG membrane in the eye disc. Interfering with the function of Rhea (i.e. the Drosophila ortholog of vertebrate talin and a key player of integrin-mediated adhesion), caused an identical stalling phenotype. These results support a key role for integrins on R-cell axons in directing R-cell axons and WG membrane to exit the eye disc.
Orr, B. O., Fetter, R. D. and Davis, G. W. (2022). Activation and expansion of presynaptic signaling foci drives presynaptic homeostatic plasticity. Neuron. PubMed ID: 36087584
Presynaptic homeostatic plasticity (PHP) adaptively regulates synaptic transmission in health and disease. Despite identification of numerous genes that are essential for PHP, a dynamic framework to explain how PHP is initiated, potentiated, and limited to achieve precise control of vesicle fusion is lacking. Utilizing both mice and Drosophila, this study demonstrates that PHP progresses through the assembly and physical expansion of presynaptic signaling foci where activated integrins biochemically converge with trans-synaptic Semaphorin2b/PlexinB signaling. Each component of the identified signaling complexes, including alpha/beta-integrin, Semaphorin2b, PlexinB, talin, and focal adhesion kinase (FAK), and their biochemical interactions, are essential for PHP. Complex integrity requires the Sema2b ligand and complex expansion includes a ~2.5-fold expansion of active-zone associated puncta composed of the actin-binding protein talin. Finally, complex pre-expansion is sufficient to accelerate the rate and extent of PHP. A working model is proposed incorporating signal convergence with dynamic molecular assemblies that instruct PHP.

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).

Talin is required to position and expand the luminal domain of the Drosophila heart tube

Fluid- and gas-transporting tubular organs are critical to metazoan development and homeostasis. Tubulogenesis involves cell polarization and morphogenesis to specify the luminal, adhesive, and basal cell domains and to establish an open lumen. This study explores a requirement for Talin, a cytoplasmic integrin adaptor, during Drosophila embryonic heart tube development. Talin marked the presumptive luminal domain and was required to orient and develop an open luminal space within the heart. Genetic analysis demonstrated that loss of zygotic or maternal-and-zygotic Talin disrupted heart cell migratory dynamics, morphogenesis, and polarity. Talin is essential for subsequent polarization of luminal determinants Slit, Robo, and Dystroglycan as well as stabilization of extracellular and intracellular integrin adhesion factors. In the absence of Talin function, mini-lumens enriched in luminal factors form in ectopic locations. Rescue experiments performed with mutant Talin transgenes suggested actin-binding was required for normal lumen formation, but not for initial heart cell polarization. The study proposes that Talin provides instructive cues to position the luminal domain and coordinate the actin cytoskeleton during Drosophila heart lumen development (Vanderploeg, 2015).

These experiments establish an essential function for the integrin adapter Talin in the assembly of the Drosophila embryonic heart. During the cardioblast (CB) migratory phase preceding tubulogenesis, Talin localizes along the CB apical surface, immediately ventral to the leading edge which extends towards the dorsal midline. As this Talin rich domain persists throughout embryonic heart assembly, eventually surrounding the lumen of the open cardiac tube, this surface is termed the pre-luminal domain. Talin is essential for the dynamic cell morphology and the leading edge features that characterise collective cardial cell migration. Furthermore, following migration, Talin is required to enclose a continuous lumen between the bilateral CB rows (Vanderploeg, 2015).

Analysis of late stage hearts in rhea zygotic mutants reveals that Talin is essential to correctly orient the CB polarity such that a continuous lumen is enclosed along the midline. In wildtype, many membrane receptors including Robo, Dg, Unc5, and Syndecan accumulate along the luminal domain. E-cadherin, Dlg, and other cell-cell adhesion factors are restricted to cell contact points immediately dorsal and ventral to the lumen and to the lateral cell domains between ipsilateral CBs. As evidenced by Robo and Dg immunolabeling experiments, the midline luminal domain is absent or, at best, is discontinuous along the midline in rhea mutant embryos. However, the Robo and Dg enriched luminal domains are not completely absent in null rhea homozygotes, but are found ectopically along lateral membranes between ipsilateral CBs. Robo's ligand, Slit, is also detected within these ectopic lumina. Similar ipsilateral Slit and Robo accumulations were observed in embryos mutant for the integrin subunit genes scab (αPS3) or mys (βPS1). Thus, the expanded Dlg-rich adhesive contact observed in rhea null embryonic hearts is consistent with a model in which integrins and Talin instruct the localization of Slit and Robo. These cues are essential to orient the lumen and to restrict the adhesive regions. In the absence of Talin, other components of the luminal structure, including Dg and the Slit-Robo complex, can self-assemble and create non-adherent luminal domains. However, proper midline positioning of the lumen requires Talin function (Vanderploeg, 2015).

Using an array of Talin transgenes previously shown to modify integrin adhesion strength and actin recruitment, this study assessed and compared the importance of these Talin-dependent processes. Binding of Talin's integrin binding site 1 (IBS1) to a membrane proximal NPxY motif on the β-integrin tail induces conformational changes within the integrin dimer, activating it and increasing the affinity for ECM ligands. Integrin activation is likely required prior to Talin IBS2 binding, an interaction which promotes a strong and stable integrin-cytoplasmic adhesome linkage. The current data indicates that either of Talin's two integrin binding sites are sufficient to promote CB morphogenesis and heart tube assembly. The ability of the heart to form in the presence of only IBS1 or IBS2 suggests that strong, long-lasting integrin-mediated adhesions are unnecessary. This idea is reinforced by the late accumulation of CAP, a protein recruited to more mature muscle adhesions. It is likely that transient adhesions are sufficient for lumenogenesis. It remains possible that an essential role for either IBS1 or IBS2 is masked by the perdurant maternal Talin in zygotic mutants. However, the functional redundancy of these domains is consistent with in vitro and in vivo studies suggesting that a subset of Talin functions can be fulfilled by either IBS domain (Vanderploeg, 2015).

Talin links integrins to the actin cytoskeleton both directly through an actin binding domain, or indirectly through recruitment of actin regulators such as Vinculin. Bond force studies of the C-terminal ABD suggest that although the ABD-actin linkage is direct, it is a weak bond which likely relies on additional direct or indirect Talin-actin linkages to form a strong and stable connection. Supporting this, TalinABD is essential for morphogenetic processes which rely on transient and dynamic integrin-actin linkages, but it is at least partially dispensable for longer-lasting adhesions which are likely stabilized by indirect Talin-actin interactions through Vinculin. The current studies demonstrate that Drosophila heart development is sensitive to disruptions in Talin's C-terminal ABD, which implicates cytoskeletal reorganization as a key process downstream of integrins during tubulogenesis. Supporting this, expression of constitutively active Diaphanous or dDAAM, formin proteins which promote actin polymerization, induced ectopic lumina similar to those that have been characterized in rhea mutants. These data are consistent with Talin promoting CB morphogenesis and lumen formation through direct, but dynamic actin linkages and suggest that formins may act downstream of Talin in apicalizing lumen formation (Vanderploeg, 2015).

To date, most studies on the Drosophila embryonic heart have focused on cell surface factors including receptors and their respective ligands; few studies have moved into the cell to establish the downstream signaling pathways involved. Insights into in vitro models suggest that polarity pathways and vesicle trafficking will be informative areas of study. For example, in the MDCK cyst model, the small GTPases Rab8a and Rab11a coordinate with the exocyst complex to deliver luminal factors to the pre-luminal initiation site. It remains to be determined whether similar exocytosis or secretion mechanisms are required for Drosophila heart lumen initiation or expansion. Furthermore, although it is unclear which classical apical polarity proteins are conserved in the Drosophila heart, epithelial and endothelial models suggest that the Cdc42-Par6-aPKC complex is a conserved master regulator of tube formation in both vertebrates and flies. Indeed, Drosophila heart tubulogenesis fails in embryos with heart specific inhibition of Cdc42 and expression of activated Cdc42 results in lateral lumina reminiscent of those characterized in rhea homozygotes. A mechanism is envisioned of heart tubulogenesis in which Talin provides instructive cues to the vesicle trafficking and polarity networks that target luminal factors and inhibit the assembly of cell-cell adhesion structures within the pre-luminal domain (Vanderploeg, 2015).

Direct binding of Talin to Rap1 is required for cell-ECM adhesion in Drosophila

Attachment of cells to the Extracellular Matrix (ECM) via integrins is essential for animal development and tissue maintenance. The cytoplasmic protein Talin is necessary for linking integrins to the cytoskeleton and its recruitment is a key step in the assembly of the adhesion complex. However, the mechanisms that regulate Talin recruitment to sites of adhesion in vivo are still not well understood. This study shows that Talin recruitment to, and maintenance at, sites of integrin-mediated adhesion requires a direct interaction between Talin and the GTPase Rap1. A mutation that blocks the direct binding of Talin to Rap1 abolished Talin recruitment to sites of adhesion and the resulting phenotype phenocopies null alleles of Talin. Moreover, this study shows that Rap1 activity modulates Talin recruitment to sites of adhesion via its direct binding to Talin. These results identify the direct Talin-Rap1 interaction as a key in vivo mechanism for controlling integrin-mediated cell-ECM adhesion (Camp, 2018).

Although Rap1 has been known as a major regulator of integrin-based adhesion for a long time, the idea that it binds to Talin directly is relatively recent. In Drosophila Rap1 plays diverse roles, and in particular is an essential regulator of cell-cell adhesion. Flies completely lacking Rap1 die during the first stages of embryonic development and exhibit severe cellular polarity defects that prevent the development of complex tissue. Importantly, Rap1 has also been previously implicated in regulating cell-ECM adhesion in Drosophila, although the mechanisms remain elusive. Initial indications of a possible direct binding of Talin and Rap1 came from solving the structure of the vertebrate talin 1 F0 domain, which revealed that the F0 domain exhibited structural similarity to the Ras-binding site in RalGDS (Goult, 2010). Subsequent studies carried out in both Dictyostelium and mammalian cell culture confirmed this direct binding and showed that it is functionally important (Plak, 2016; Zhu, 2017). Two key functional observations have emerged from these studies: first, that the binding of Talin to Rap1 is low affinity and, second, that the binding of Talin to Rap1 regulates Talin recruitment to the membrane (Plak, 2016; Zhu, 2017). The current work confirms and builds upon these previous observations. The phenotype observed when a mutation was introduced in Talin that blocks Rap1 binding is indistinguishable from that observed in a null mutation that completely abolishes Talin function. FRAP and localization studies suggests that the strong phenotype caused by loss of direct binding of Talin to Rap1 can be explained by a disruption in the ability of the mutant Talin to be localized to and/or be maintained at sites of integrin-mediated adhesion. This result is somewhat surprising because Talin has multiple means of localizing to sites of integrin-mediated adhesion, including two integrin-binding sites, a binding site for the RAP1-binding scaffolding protein RIAM, a phosphatidylinositol 4,5-bisphosphate (PIP2) interaction domain, as well as multiple other domains that interact with other components of the adhesion complex. It thus appears that, at least in certain contexts, the interaction with Rap1, although weak in nature, is of particular importance for controlling Talin localization. Previous work has suggested that the direct interaction between Talin and Rap1 is strengthened when Rap1 is anchored to the membrane (Zhu, 2017). In this model, Rap1 localization to sites of adhesion creates a microenvironment that favours the recruitment and/or maintenance of Talin (Zhu, 2017). This model is very compatible with the current findings and helps explain earlier results that defined a role for Rap1 in regulating integrin-mediated adhesion in flies (Camp, 2018).

The direct binding of Rap1 to Talin also fits very well with earlier work on Talin autoinhibition (Ellis, 2013). Previous work showed that Talin that is unable to undergo autoinhibition localizes to the membrane independently of the presence of RIAM (Ellis, 2013). This suggested the existence of a RIAM-independent mechanism for Talin localization. Intriguingly, previous studies show that the F2F3 domain of Talin contains a conserved RIAM-binding site. The results show that although the F2F3 domain of Talin localizes to sites of adhesion, it does so less efficiently than the full Talin head domain (containing F0-F3). Furthermore, in contrast to the full-length Talin head construct, the recruitment of a construct containing only the F2F3 domain was not efficiently modulated by the presence of constitutively activated Rap1. This indicates that the main way Rap1 controls the recruitment of the Talin head domain to sites of integrin-mediated adhesion is through direct binding to Talin rather than indirect binding through RIAM. Support for these conclusions comes from studies in mice that showed that RIAM was dispensable in most tissues for Talin localization and integrin activation. Therefore, it appears that in flies, and possibly, in some tissues in mice, the direct interaction of Talin with Rap is both necessary and sufficient for recruitment and/or maintenance of Talin at sites of integrin-mediated adhesion, and that this applies to both autoinhibited and non-autoinhibited Talin (Camp, 2018).

The data suggests that a Talin mutant that is unable to bind directly to Rap1 is transcribed, translated and folds normally, but is unable to function. While a model is favored wherein the strong phenotype that was observed is due to an inability to localize and/or maintain Talin at sites of adhesion, several other alternative hypotheses cannot be discounted. For example, it could be that, in addition to disrupting the interaction of Talin with Rap1, the K17 mutation also disrupts the interaction of Talin with proteins other than Rap1. Possible candidates for such additional interactions with the Talin F0 domain are other Ras GTPases encoded by the Drosophila genome. However, the possible roles of fly GTPases were explored in the context of a genome wide analysis that analysed, among other phenotypes, disruption to integrin-based myotendinous junctions. This analysis failed to identify a role for additional Ras GTPases in integrin-mediated muscle tendon attachment. Additionally, while the experiments show direct F0-Rap1 interactions in vitro they do not address the specificity or selectivity of the interaction. The overall weak binding between Talin F0 and Rap1 makes it difficult to illustrate this interaction in vivo, which leaves the possibility of indirect binding or that additional, partially redundant, factors are involved. Furthermore, in previous work it was shown that making a 'headless' version of Talin by deleting the entire Talin head (residues 1-448), while leaving the rest of Talin intact results in a Talin protein that is completely non-functional, but that can still partially localize to sites of adhesion (x 2014). Given this result, it is curious that mutating the single K17 residue in the Talin head completely abolishes Talin localization. One possible explanation is that the headless version of Talin was expressed from a ubiquitous promoter in an exogenous rescue construct, in contrast to the CRISPR approach used in this study. Another possible explanation is that the headless Talin was tagged with GFP, and was detected using an extremely sensitive GFP-specific antibody in contrast to the Talin-specific antibody used to detect the K17 mutant Talin in the present study. Nonetheless, these results hint at the complicated network of positive and negative reinforcement cues that operate on Talin to regulate its localization to the membrane (Camp, 2018).

It remains to be fully established whether the important role of direct binding between Talin and Rap1 is conserved in vertebrates. The higher complexity of integrin-based adhesions in vertebrates provides alternative regulatory mechanisms that can mask the sort of dramatic phenotypic effects observed in disruptions of the simpler integrin-based adhesions found in the fly. Consistent with this idea is the recent analysis of mice containing a mutation in Talin designed to block the interaction of the F0 domain of Talin with Rap1 (Lagarrigue, 2018). That work shows that, at least in the context of blood platelets, the direct interaction between the F0 domain and Rap1 is not essential for integrin activation. This suggests two possible differences between the fly and vertebrate integrin-based adhesions, the first is that, in vertebrates, Talin can be recruited to the membrane by multiple, Rap1-independent, mechanisms, and the second is that, in vertebrates, Rap1 binds to Talin directly through another interaction that does not involve the F0 domain and which is not conserved in flies. Nonetheless, by showing in the fly that Rap1 binding is essential for Talin recruitment to sites of adhesion this work raises the possibility that, at least in some contexts in vertebrates, the direct binding of Talin to Rap1 may prove to be functionally important (Camp, 2018).

Slik phosphorylation of talin T152 is crucial for proper talin recruitment and maintenance of muscle attachment in Drosophila
Talin is the major scaffold protein linking integrin receptors with the actin cytoskeleton. In Drosophila, extended talin generates a stable link between the sarcomeric cytoskeleton and the tendon matrix at muscle attachment sites. This study identified phosphorylation sites on Drosophila talin by mass spectrometry. Talin is phosphorylated in late embryogenesis when muscles differentiate, especially on T152 in the exposed loop of the F1 domain of the talin head. Localization of talin-T150/T152A is reduced at muscle attachment sites and can only partially rescue muscle attachment compared to wild type talin. Slik was identified as the kinase phosphorylating talin at T152. Slik localizes to muscle attachment sites, and the absence of Slik reduces the localization of talin at muscle attachment sites causing phenotypes similar to talin-T150/T152A. Thus, these results demonstrate that talin phosphorylation by Slik plays an important role in fine-tuning talin recruitment to integrin adhesion sites and maintaining muscle attachment (Katzemich, 2019).

Phosphorylation of the Talin FERM domain by Slik as being important for Talin function in muscles. This pathway is likely conserved in vertebrates, because SLK and talin colocalize at focal adhesions and a conditional Slk knockout in skeletal muscles results in progressive myopathy. In platelets, the high stoichiometry phosphorylation sites of Talin are T144 and T150. The effect of their phosphorylation is somewhat inconclusive, but it appears that T144/T150A mutations in tissue culture reduce cell adhesion and increase focal adhesion turnover. A recent structural study indicates that the unstructured F1 loop does not interact with positively charged membrane phospholipids, suggesting that F1 loop phosphorylation should not disrupt membrane recruitment. This is consistent with the full rescue observed with T152E. Talin-E1777A, a mutant keeping Talin always in the extended, active conformation, fully rescues muscle detachment, and localizes more strongly to muscle attachment sites than does wild-type Talin. A multi-step mechanism of Talin activation is proposed, in which phosphorylation of threonine 150/152 is one step contributing to Talin activation and separation of the head and rod domains of Talin. Although the detailed mechanism and the sequence of events remain to be uncovered, this work identifies the first kinase involved in Talin F1 loop phosphorylation and demonstrates that this phosphorylation is crucial for the maintenance of muscle attachment (Katzemich, 2019).


cDNA clone length - 9347 bp

Bases in 5' UTR - 225

Exons - 15

Bases in 3' UTR - 633


Amino Acids - 2836

Structural Domains

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).

Talin: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 11 May 2022

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