Differences in elasticity of vinculin-deficient F9 cells measured by magnetometry and atomic force microscopy.
We have investigated a mouse F9 embryonic carcinoma cell line, in which both vinculin genes were inactivated by homologous recombination, that exhibits defective adhesion and spreading [Coll et al. (1995) Proc. Natl. Acad. Sci. USA 92, 9161-9165]. Using a magnetometer and RGD-coated magnetic microbeads, we measured the local effect of loss and replacement of vinculin on mechanical force transfer across integrins. Vinculin-deficient F9Vin(-/-) cells showed a 21% difference in relative stiffness compared to wild-type cells. This was restored to near wild-type levels after transfection and constitutive expression of increasing amounts of vinculin into F9Vin(-/-) cells. In contrast, the transfection of vinculin constructs deficient in amino acids 1-288 (containing the talin- and alpha-actinin-binding site) or substituting tyrosine for phenylalanine (phosphorylation site, amino acid 822) in F9Vin(-/-) cells resulted in partial restoration of stiffness. Using atomic force microscopy to map the relative elasticity of entire F9 cells by 128 x 128 (n = 16,384) force scans, we observed a correlation with magnetometer measurements. These findings suggest that vinculin may promote cell adhesions and spreading by stabilizing focal adhesions and transferring mechanical stresses that drive cytoskeletal remodeling, thereby affecting the elastic properties of the cell.
Goldmann
W H
WH
Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Charlestown 02129, USA. goldman@helix.mgh
Galneder
R
R
Ludwig
M
M
Xu
W
W
Adamson
E D
ED
Wang
N
N
Ezzell
R M
RM
D000818:Animals; D018236:Carcinoma, Embryonal / Q000473:pathology*; D002448:Cell Adhesion; D003599:Cytoskeleton / Q000502:physiology; D004548:Elasticity; D016326:Extracellular Matrix Proteins / Q000502:physiology; D018390:Gene Targeting; D046911:Macromolecular Substances; D008280:Magnetics; D051379:Mice; D018625:Microscopy, Atomic Force; D008863:Microspheres; D009842:Oligopeptides; D013314:Stress, Mechanical; D014407:Tumor Cells, Cultured; D016596:Vinculin / Q000172:deficiency / Q000235:genetics / Q000502:physiology*
D016326:Extracellular Matrix Proteins; D046911:Macromolecular Substances; D009842:Oligopeptides; D016596:Vinculin; C047981:arginyl-glycyl-aspartic acid