Anchorage of vinculin to lipid membranes influences cell mechanical properties.
The focal adhesion protein vinculin (1066 residues) can be separated into a 95-kDa head and a 30-kDa tail domain. Vinculin's lipid binding sites localized on the tail, helix 3 (residues 944-978) and the unstructured C-terminal arm (residues 1052-1066, the so-called lipid anchor), influence focal adhesion turnover and are important for cell migration and adhesion. Using magnetic tweezers, we characterized the cell mechanical behavior in mouse embryonic fibroblast (MEF)-vin(-/-) cells transfected with EGFP-linked-vinculin deficient of the lipid anchor (vinDeltaC, residues 1-1051). MEF-vinDeltaC cells incubated with fibronectin-coated paramagnetic beads were less stiff, and more beads detached during these experiments compared to MEF-rescue cells. Cells expressing vinDeltaC formed fewer focal contacts as determined by confocal microscopy. Two-dimensional traction measurements showed that MEF-vinDeltaC cells generate less force compared to rescue cells. Attenuated traction forces were also found in cells that expressed vinculin with point mutations (R1060 and K1061 to Q) of the lipid anchor that impaired lipid binding. However, traction generation was not diminished in cells that expressed vinculin with impaired lipid binding caused by point mutations on helix 3. Mutating the src-phosphorylation site (Y1065 to F) resulted in reduced traction generation. These observations show that both the lipid binding and the src-phosphorylation of vinculin's C-terminus are important for cell mechanical behavior.
Diez
Gerold
G
Center for Medical Physics and Technology, Biophysics Group, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany.
Kollmannsberger
Philip
P
Mierke
Claudia T
CT
Koch
Thorsten M
TM
Vali
Hojatollah
H
Fabry
Ben
B
Goldmann
Wolfgang H
WH
D000818:Animals; D001696:Biomechanical Phenomena; D002448:Cell Adhesion; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D050356:Lipid Metabolism; D008280:Magnetics; D051379:Mice; D008853:Microscopy; D010766:Phosphorylation; D011485:Protein Binding; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000172:deficiency / Q000378:metabolism; D014162:Transfection; D016596:Vinculin / Q000172:deficiency / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism
D005353:Fibronectins; D011993:Recombinant Fusion Proteins; D016596:Vinculin; D019061:src-Family Kinases