New isoform-specific monoclonal antibodies reveal different sub-cellular localisations for talin1 and talin2.
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.
Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK.
Uta
U
Praekelt
Petra M
PM
Kopp
Kerstin
K
Rehm
Stefan
S
Linder
Neil
N
Bate
Bipin
B
Patel
Emmanuel
E
Debrand
Ana Maria
AM
Manso
Robert S
RS
Ross
Franceso
F
Conti
Ming-Zhi
MZ
Zhang
Raymond C
RC
Harris
Roy
R
Zent
David R
DR
Critchley
Susan J
SJ
Monkley
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D005353:Fibronectins / Q000378:metabolism*; D022001:Focal Adhesions / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006801:Humans; D008264:Macrophages / Q000648:ultrastructure*; D051379:Mice; D008807:Mice, Inbred BALB C; D041681:NIH 3T3 Cells; D020033:Protein Isoforms / Q000032:analysis* / Q000378:metabolism; D034741:RNA, Small Interfering; D051381:Rats; D016608:Talin / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D005353:Fibronectins; D020033:Protein Isoforms; D034741:RNA, Small Interfering; C518833:TLN2 protein, mouse; D016608:Talin; C496077:Tln1 protein, mouse