Analysis of EST data produced primarily by Nori Satoh and colleagues of Kyoto University  has shown that alternative splicing of talin pre-mRNA leads to the production of two proteins with different C-termini in C. intestinalis. The variable distributions of Talin-a and Talin-b in different cDNA libraries suggest that Talin-a and Talin-b are differentially expressed during C. intestinalis development. We have also shown that the variant I/LWEQ modules of Talin-a and Talin-b have different biological properties: Talin-b has the higher affinity for F-actin and preferentially labels actin stress fibers, while Talin-a is targeted to focal adhesions. Therefore, alternative processing is a mechanism for producing multiple talin proteins, with distinct activities associated with a critical structural element, in these nonvertebrate chordates, even though the C. intestinalis genome contains only one talin gene.
Alternative splicing is a widespread mechanism for expanding the proteome. In humans, 30–65% of all gene products are subject to alternative splicing, and similar levels are also seen in other organisms, including the mouse, fruitfly, and nematode . Alternative splicing frequently results in changes in protein structure that alter protein-protein interactions. For example, alternative splicing of the non-erythroid protein 4.1R produces variants that alter the efficiency of the interaction with the spectrin/actin complex by a factor of two . This is similar to differences we have identified in the C. intestinalis talin alternative splice variants, where the Talin-b I/LWEQ module interacts with F-actin with a four-fold higher affinity than that of Talin-a. The higher affinity for F-actin may explain the preferential localization of the Talin-b I/LWEQ module to actin stress fibers in cells.
Alternative splicing can also alter subcellular targeting determinants of the resultant proteins . HeLa cells represent a heterologous system for the study of tunicate talins in vivo, but we have recently used them to determine that the C-terminal I/LWEQ module of Talin1 contains a focal adhesion targeting determinant . We have shown here that the Talin-a I/LWEQ module, but not that of Talin-b, is targeted to focal adhesions in HeLa cells. Although focal adhesions have not yet been directly observed in C. intestinalis cells, the C. intestinalis genome contains genes for several characteristic focal adhesion components that were previously thought to be restricted to vertebrates. These include at least 14 α-integrins, 5 β-integrins, focal adhesion kinase, vinculin, and α-actinin . Vinculin and β-integrin are well-characterized Talin1 binding partners. Given the presence of these classical focal adhesion proteins in C. intestinalis, our results suggest that Talin-a is likely to function similarly to mammalian Talin1 as an adhesion complex component during C. intestinalis development. The lack of a focal adhesion-targeting determinant in the C-terminus of Talin-b may indicate that this protein is a component of other adhesion complexes in the organism, as we have seen with mammalian Talin2. Talin2 is not targeted to focal adhesions in undifferentiated cells but is preferentially targeted to costameres and intercalated disks, which are stable adhesions in differentiated striated muscle .
The presence of two related, but ancient and highly divergent talins (30% identical) with different physiological roles in D. discoideum [4–6] supports the hypothesis that gene duplication, followed by neo-functionalization, produced multiple talins that are required for cell adhesion during cell differentiation [3, 29]. A similar explanation may also account for the maintenance of Talin1 and Talin2 in vertebrates since duplication of the ancestral animal talin early in the chordate lineage, more than 450 million years ago . In support of this hypothesis regarding vertebrate talins, several recent studies have shown that these distinct proteins indeed have different roles. Talin1 and Talin2 interact with different proteins [3, 30, 31] and have different affinities for their common partner F-actin . Talin1 is required for mammalian embryogenesis, probably as an essential component of adhesion complexes required for cell motility during gastrulation . Talin2 is unable to complement this lethal phenotype. We have recently shown that the adult tissue distributions of mammalian Talin1 and Talin2 also vary, with Talin1 being the more widely expressed isoform . Talin2 is most abundant in brain and muscle. We have shown that Talin2 is induced during muscle differentiation along with other muscle-specific proteins such as archvillin, integrin-β1D, and metavinculin, which is an alternative splice variant of vinculin . As expected, Talin1 is a component of focal adhesions in undifferentiated myoblasts, but Talin2 is not found in focal adhesions of these cells or other mammalian cells. Talin2 is instead a component of stable adhesion assemblies such as costameres and intercalated disks in mature striated muscle . Taken together, these studies indicate that vertebrate Talin1 and Talin2 are differentially expressed during cell differentiation and organismal development and that they function as non-redundant components of distinct adhesion complexes in different cells and tissues. Thus, in both D. discoideum and vertebrates, multiple talins are involved in talin functions.
Development in C. intestinalis is a complex, multistage process. Following fertilization and subsequent embryogenesis, a free-swimming tadpole larva containing a notochord, nervous system, and musculature settles upon a solid substrate and metamorphoses into a sessile, filter-feeding organism. The adult contains organs common to other chordates, including a digestive system, heart, and nervous system . C. intestinalis has only one talin gene, but the existence of the splice variants Talin-a and Talin-b, which vary in the important C-terminal I/LWEQ module , further supports the emerging paradigm that talin function is due to the actions of distinct proteins, which are expressed at different times and in different places during chordate development [3, 10]. The results presented here suggest that alternative splicing is the means by which two talins with different roles in cell adhesion, and perhaps cell differentiation, are produced from one talin gene in C. intestinalis. Thus, this mechanism may bridge the one-talin bottleneck present in invertebrates between the multicellular amoebozoan D. discoideum  and vertebrates . Further studies at the cellular and molecular levels will identify when and where Talin-a and Talin-b are produced in C. intestinalis, and how they contribute to the assembly and function of adhesion complexes during development of this model organism.