title: Cooperative Vinculin Binding to Talin Mapped by Time-Resolved Super Resolution Microscopy.
abstract: The dimeric focal adhesion protein talin contains up to 22 cryptic vinculin binding sites that are exposed by unfolding. Using a novel method to monitor the in situ dynamics of the talin dimer stretch, we find that in contrast to several prevalent talin dimer models the integrin-binding talin N-termini are separated by 162 ± 44 nm on average whereas as expected the C-terminal dimerization domains colocalize and are mobile. Using vinculin tagged by DHFR-TMP Atto655 label, we found that optimal vinculin and vinculin head binding occurred when talin was stretched to 180 nm, while the controls did not bind to talin. Surprisingly, multiple vinculins bound within a single second in narrowly localized regions of the talin rod during stretching. We suggest that talin stretches as an antiparallel dimer and that activates vinculin binding in a cooperative manner, consistent with the stabilization of folded talin by other binding proteins.
authors.affiliation: Mechanobiology Institute, National University of Singapore , Singapore 117411, Singapore.
authors.forename: Xian
authors.initials: X
authors.lastname: Hu
authors.affiliation: Department of Chemistry, Columbia University , New York, New York 10027, United States.
authors.forename: Chaoran
authors.initials: C
authors.lastname: Jing
authors.affiliation: Mechanobiology Institute, National University of Singapore , Singapore 117411, Singapore.
authors.forename: Xiaochun
authors.initials: X
authors.lastname: Xu
authors.affiliation: Mechanobiology Institute, National University of Singapore , Singapore 117411, Singapore.
authors.forename: Naotaka
authors.initials: N
authors.lastname: Nakazawa
authors.affiliation: Department of Chemistry, Columbia University , New York, New York 10027, United States.
authors.forename: Virginia W
authors.initials: VW
authors.lastname: Cornish
authors.affiliation: Mechanobiology Institute, National University of Singapore , Singapore 117411, Singapore.
authors.forename: Felix M
authors.initials: FM
authors.lastname: Margadant
authors.affiliation: Mechanobiology Institute, National University of Singapore , Singapore 117411, Singapore.|Department of Biological Sciences, Columbia University , New York, New York 10027, United States.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Sheetz
keywords: Talin; focal adhesion; localization microscopy; protein recruitment; single molecule tracking; vinculin