title: Integrin-associated complexes form hierarchically with variable stoichiometry in nascent adhesions.
abstract: BACKGROUND
  A complex network of putative molecular interactions underlies the architecture and function of cell-matrix adhesions. Most of these interactions are implicated from coimmunoprecipitation studies using expressed components, but few have been demonstrated or characterized functionally in living cells.
  
  
  RESULTS
  We introduce fluorescence fluctuation methods to determine, at high spatial and temporal resolution, "when" and "where" molecular complexes form and their stoichiometry in nascent adhesions (NAs). We focus on integrin-associated molecules implicated in integrin activation and in the integrin-actin linkage in NAs and show that these molecules form integrin-containing complexes hierarchically within the adhesion itself. Integrin and kindlin reside in a molecular complex as soon as adhesions are visible; talin, although also present early, associates with the integrin-kindlin complex only after NAs have formed and in response to myosin II activity. Furthermore, talin and vinculin association precedes the formation of the integrin-talin complex. Finally, α-actinin enters NAs periodically and in clusters that transiently associate with integrins. The absolute number and stoichiometry of these molecules varies among the molecules studied and changes as adhesions mature.
  
  
  CONCLUSIONS
  These observations suggest a working model for NA assembly whereby transient α-actinin-integrin complexes help nucleate NAs within the lamellipodium. Subsequently, integrin complexes containing kindlin, but not talin, emerge. Once NAs have formed, myosin II activity promotes talin association with the integrin-kindlin complex in a stoichiometry consistent with each talin molecule linking two integrin-kindlin complexes.
authors.affiliation: Department of Cell Biology, University of Virginia, Charlottesville, VA 22908, USA. Electronic address: ab8su@virginia.edu.
authors.forename: Alexia I
authors.initials: AI
authors.lastname: Bachir
authors.affiliation: Department of Cell Biology, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Zareno
authors.affiliation: Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Konstadinos
authors.initials: K
authors.lastname: Moissoglu
authors.affiliation: Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA.
authors.forename: Edward F
authors.initials: EF
authors.lastname: Plow
authors.affiliation: Laboratory of Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, Irvine, CA 92697, USA.
authors.forename: Enrico
authors.initials: E
authors.lastname: Gratton
authors.affiliation: Department of Cell Biology, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Alan R
authors.initials: AR
authors.lastname: Horwitz
mesh_terms: D000818:Animals; D016466:CHO Cells; D021982:Cell-Matrix Junctions / Q000378:metabolism*; D003412:Cricetulus; D016023:Integrins / Q000378:metabolism*
chemical_list: D016023:Integrins
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