Talin determines the nanoscale architecture of focal adhesions.
Insight into how molecular machines perform their biological functions depends on knowledge of the spatial organization of the components, their connectivity, geometry, and organizational hierarchy. However, these parameters are difficult to determine in multicomponent assemblies such as integrin-based focal adhesions (FAs). We have previously applied 3D superresolution fluorescence microscopy to probe the spatial organization of major FA components, observing a nanoscale stratification of proteins between integrins and the actin cytoskeleton. Here we combine superresolution imaging techniques with a protein engineering approach to investigate how such nanoscale architecture arises. We demonstrate that talin plays a key structural role in regulating the nanoscale architecture of FAs, akin to a molecular ruler. Talin diagonally spans the FA core, with its N terminus at the membrane and C terminus demarcating the FA/stress fiber interface. In contrast, vinculin is found to be dispensable for specification of FA nanoscale architecture. Recombinant analogs of talin with modified lengths recapitulated its polarized orientation but altered the FA/stress fiber interface in a linear manner, consistent with its modular structure, and implicating the integrin-talin-actin complex as the primary mechanical linkage in FAs. Talin was found to be ∼97 nm in length and oriented at ∼15° relative to the plasma membrane. Our results identify talin as the primary determinant of FA nanoscale organization and suggest how multiple cellular forces may be integrated at adhesion sites.
Mechanobiology Institute, Singapore, National University of Singapore, Republic of Singapore 117411;
Jaron
J
Liu
Mechanobiology Institute, Singapore, National University of Singapore, Republic of Singapore 117411;
Yilin
Y
Wang
Mechanobiology Institute, Singapore, National University of Singapore, Republic of Singapore 117411;
Wah Ing
WI
Goh
Mechanobiology Institute, Singapore, National University of Singapore, Republic of Singapore 117411;
Honzhen
H
Goh
National High Magnetic Field Laboratory, The Florida State University, Tallahassee, FL 32306;
Michelle A
MA
Baird
Mechanobiology Institute, Singapore, National University of Singapore, Republic of Singapore 117411;
Svenja
S
Ruehland
Mechanobiology Institute, Singapore, National University of Singapore, Republic of Singapore 117411;
Shijia
S
Teo
Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom;
Neil
N
Bate
Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom;
David R
DR
Critchley
National High Magnetic Field Laboratory, The Florida State University, Tallahassee, FL 32306; Department of Biological Sciences, The Florida State University, Tallahassee, FL 32306;
Michael W
MW
Davidson
Mechanobiology Institute, Singapore, National University of Singapore, Republic of Singapore 117411; Department of Biomedical Engineering, National University of Singapore, Republic of Singapore 117411 biekp@nus.edu.sg.
Pakorn
http://orcid.org/0000-0001-8765-3861
P
Kanchanawong
D022001:Focal Adhesions / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence; D049329:Nanostructures; D016608:Talin / Q000502:physiology*
D016608:Talin
focal adhesions; mechanobiology; nanoscale architecture; superresolution microscopy; talin