New isoform-specific monoclonal antibodies reveal different sub-cellular localisations for talin1 and talin2

Abstract

Talins are adaptor proteins that connect the integrin family of cell adhesion receptors to cytoskeletal actin. Vertebrates express two closely related talins encoded by separate genes, and while it is well established that talin1 plays a key role in cell adhesion and spreading, little is known about the role of talin2. To facilitate such studies, we report the characterisation of 4 new isoform-specific talin mouse monoclonal antibodies that work in Western blotting, immuno-precipitation, immuno-fluorescence and immuno-histochemistry. Using these antibodies, we show that talin1 and talin2 do not form heterodimers, and that they are differentially localised within the cell. Talin1 was concentrated in peripheral focal adhesions while talin2 was observed in both focal and fibrillar adhesions, and knock-down of talin2 compromised fibronectin fibrillogenesis. Although differentiated human macrophages express both isoforms, only talin1 showed discrete staining and was localised to the ring structure of podosomes. However, siRNA-mediated knock-down of macrophage talin2 led to a significant reduction in podosomal matrix degradation. We have also used the antibodies to localise each isoform in tissue sections using both cryostat and paraffin-embedded material. In skeletal muscle talin2 was localised to both myotendinous junctions and costameres while talin1 was restricted to the former structure. In contrast, both isoforms co-localised in kidney with staining of the glomerulus, and the tubular epithelial and interstitial cells of the cortex and medulla. We anticipate that these antibodies will form a valuable resource for future studies on the function of the two major talin isoforms.

Introduction

Cell adhesion to the extracellular matrix (ECM) plays a key role in the migration, proliferation and differentiation of animal cells, and their organisation into tissues and organs during embryonic development. Several types of cell–ECM junctions have been characterised in cultured cells (Dubash et al., 2009) including focal complexes formed at the leading edge of migratory cells, which in turn mature into larger more elongated focal adhesions (FA). Fibrillar adhesions (FB) are found in the central area of the cell and are associated with the fibronectin fibrillogenesis (Cukierman et al., 2001), while podosomes and invadopodia are more specialised adhesion structures that are only found in certain cell types (Block et al., 2008). All the above cell–ECM junctions share the same general architecture and composition, i.e. the extracellular domains of the integrin family of α/β hetero-dimeric trans-membrane proteins are bound to ECM proteins, while the short cytoplasmic tails of the integrin β-subunits are linked to the actin cytoskeleton via a variety of adaptor proteins (Brakebusch and Fassler, 2003, Legate and Fassler, 2009). One such adaptor is talin (∼270 kDa, ∼2540 amino acids) which binds both β-integrin tails and F-actin (Critchley, 2009), and also modulates the affinity of integrin for ligands (Anthis and Campbell, 2011, Shattil et al., 2010). Talin consists of an N-terminal head containing an atypical FERM domain (Elliott et al., 2010, Goult et al., 2010) that binds β-integrin tails (Anthis et al., 2009, Calderwood et al., 1999, Wegener et al., 2007) coupled to an elongated flexible rod with a second integrin binding site (Gingras et al., 2009, Moes et al., 2007), at least two actin binding sites (Gingras et al., 2008, Hemmings et al., 1996), and multiple binding sites for the cytoskeletal protein vinculin (Gingras et al., 2005). Much of talin in the cell is thought to exist in an autoinhibited cytoplasmic form due to intramolecular interactions between the head and rod (Goksoy et al., 2008, Goult et al., 2009), and both Rap1/RIAM (Han et al., 2006, Lee et al., 2009) and PIP2 (Goksoy et al., 2008, Martel et al., 2001) have been implicated in talin activation.

In vertebrates there are two talin genes, Tln1 and Tln2, which encode very similar proteins (74% amino acid sequence identity) (Debrand et al., 2009, Monkley et al., 2001). Tln2 appears to be the ancestral gene with Tln1 arising by gene duplication early in the chordate lineage (Senetar and McCann, 2005). However, the role of the two major talin isoforms remains unclear. Knockout of Tln1 is embryonic lethal at gastrulation (Monkley et al., 2000) while Tln2 knockout mice are viable and fertile (Chen and Lo, 2005), although they have a mildly dystrophic phenotype that is more severe than that arising from muscle-specific knockout of Tln1 (Conti et al., 2008, Conti et al., 2009). Interestingly, talin2 has a much higher affinity for the cytoplasmic tail of β1D-integrin (Anthis et al., 2010), a splice variant that is localised with talin2 in the myotendinous junction of striated muscle. This suggests a model in which the tight binding of talin2 to β1D-integrin is designed to withstand the high forces exerted on the myotendinous junction in vivo. Loss of both talin1 and talin2 from muscle leads to severe defects in myogenesis and is perinatal lethal, indicating that the two isoforms have overlapping but non-redundant functions in muscle (Conti et al., 2009).

Further progress in understanding the function of talin1 and talin2 has been restricted by the lack of antibodies that are specific for each isoform. Many of the commonly used commercial antibodies, e.g. 8d4 and TD77 (Sigma) recognise both isoforms, while the only talin1-specific antibody (TA205) detects human but not mouse talin1 (Bolton et al., 1997). Here we characterise four new isoform-specific monoclonal antibodies (Mabs) that detect either talin1 or talin2 from a range of species, and that work in Western blotting, immuno-precipitation, immuno-fluorescence and immuno-histochemistry. We have used these antibodies to analyse the sub-cellular localisation and tissue distribution of both isoforms, and show for the first time that in NIH3T3 cells, smooth muscle cells, mouse embryo fibroblasts and macrophages, the sub-cellular localisation of these two very similar proteins is quite distinct.