A Structural Model for Vinculin Insertion into PIP2-Containing Membranes and the Effect of Insertion on Vinculin Activation and Localization

Peter M Thompson; Srinivas Ramachandran; Lindsay B Case; Caitlin E Tolbert; Arpit Tandon; Mihir Pershad; Nikolay V Dokholyan; Clare M Waterman; Sharon L Campbell

doi:10.1016/j.str.2016.12.002

A Structural Model for Vinculin Insertion into PIP₂-Containing Membranes and the Effect of Insertion on Vinculin Activation and Localization

Structure. 2017 Feb 7;25(2):264-275. doi: 10.1016/j.str.2016.12.002. Epub 2017 Jan 12.

Authors

Peter M Thompson¹, Srinivas Ramachandran¹, Lindsay B Case², Caitlin E Tolbert³, Arpit Tandon¹, Mihir Pershad⁴, Nikolay V Dokholyan⁵, Clare M Waterman², Sharon L Campbell⁶

Affiliations

¹ Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
² Cell Biology and Physiology Center, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
³ Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
⁴ Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
⁵ Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
⁶ Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address: campbesl@med.unc.edu.

Abstract

Vinculin, a scaffolding protein that localizes to focal adhesions (FAs) and adherens junctions, links the actin cytoskeleton to the adhesive super-structure. While vinculin binds to a number of cytoskeletal proteins, it can also associate with phosphatidylinositol 4,5-bisphosphate (PIP₂) to drive membrane association. To generate a structural model for PIP₂-dependent interaction of vinculin with the lipid bilayer, we conducted lipid-association, nuclear magnetic resonance, and computational modeling experiments. We find that two basic patches on the vinculin tail drive membrane association: the basic collar specifically recognizes PIP₂, while the basic ladder drives association with the lipid bilayer. Vinculin mutants with defects in PIP₂-dependent liposome association were then expressed in vinculin knockout murine embryonic fibroblasts. Results from these analyses indicate that PIP₂ binding is not required for localization of vinculin to FAs or FA strengthening, but is required for vinculin activation and turnover at FAs to promote its association with the force transduction FA nanodomain.

Keywords: PIP2; focal adhesion; iPALM; lipid bilayer; molecular dynamics; nuclear magnetic resonance; vinculin.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Actin Cytoskeleton / genetics
Actin Cytoskeleton / metabolism*
Actins / genetics
Actins / metabolism*
Amino Acid Motifs
Animals
Binding Sites
Embryo, Mammalian
Fibroblasts / metabolism
Fibroblasts / ultrastructure
Focal Adhesions / metabolism*
Focal Adhesions / ultrastructure
Gene Expression
Hydrophobic and Hydrophilic Interactions
Lipid Bilayers / chemistry*
Lipid Bilayers / metabolism
Mechanotransduction, Cellular
Mice
Molecular Docking Simulation
Molecular Dynamics Simulation
Mutation
Nuclear Magnetic Resonance, Biomolecular
Phosphatidylinositol 4,5-Diphosphate / chemistry*
Phosphatidylinositol 4,5-Diphosphate / metabolism
Protein Binding
Protein Conformation, alpha-Helical
Protein Interaction Domains and Motifs
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Recombinant Proteins / metabolism
Thermodynamics
Vinculin / chemistry*
Vinculin / genetics
Vinculin / metabolism

Substances

Actins
Lipid Bilayers
Phosphatidylinositol 4,5-Diphosphate
Recombinant Proteins
Vinculin

Abstract

Publication types

MeSH terms

Substances

Grants and funding