title: Molecular mechanism of vinculin activation and nanoscale spatial organization in focal adhesions.
abstract: Focal adhesions (FAs) link the extracellular matrix to the actin cytoskeleton to mediate cell adhesion, migration, mechanosensing and signalling. FAs have conserved nanoscale protein organization, suggesting that the position of proteins within FAs regulates their activity and function. Vinculin binds different FA proteins to mediate distinct cellular functions, but how vinculin's interactions are spatiotemporally organized within FAs is unknown. Using interferometric photoactivation localization super-resolution microscopy to assay vinculin nanoscale localization and a FRET biosensor to assay vinculin conformation, we found that upward repositioning within the FA during FA maturation facilitates vinculin activation and mechanical reinforcement of FAs. Inactive vinculin localizes to the lower integrin signalling layer in FAs by binding to phospho-paxillin. Talin binding activates vinculin and targets active vinculin higher in FAs where vinculin can engage retrograde actin flow. Thus, specific protein interactions are spatially segregated within FAs at the nanoscale to regulate vinculin activation and function.
authors.affiliation: Cell Biology and Physiology Center, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Lindsay B
authors.initials: LB
authors.lastname: Case
authors.affiliation: National High Magnetic Field Laboratory and Department of Biological Science, The Florida State University, Tallahassee, Florida 32310, USA.
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Baird
authors.affiliation: Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia 20147, USA.
authors.forename: Gleb
authors.initials: G
authors.lastname: Shtengel
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA.
authors.forename: Sharon L
authors.initials: SL
authors.lastname: Campbell
authors.affiliation: Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia 20147, USA.
authors.forename: Harald F
authors.initials: HF
authors.lastname: Hess
authors.affiliation: National High Magnetic Field Laboratory and Department of Biological Science, The Florida State University, Tallahassee, Florida 32310, USA.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Davidson
authors.affiliation: Cell Biology and Physiology Center, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Clare M
authors.initials: CM
authors.lastname: Waterman
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D031541:Fluorescence Resonance Energy Transfer; D022001:Focal Adhesions / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016023:Integrins / Q000737:chemistry / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D008958:Models, Molecular; D009154:Mutation; D049329:Nanostructures; D036103:Nanotechnology / Q000379:methods*; D051419:Paxillin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D016608:Talin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016596:Vinculin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000199:Actins; D016023:Integrins; D008164:Luminescent Proteins; D051419:Paxillin; D016608:Talin; D016596:Vinculin