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.

[1]  M. Radmacher,et al.  In vitro activation of human platelets triggered and probed by atomic force microscopy. , 1993, Experimental cell research.

[2]  J. Bereiter-Hahn.  Cytomechanics and Biochemistry , 1991 .

[3]  Steen Rasmussen,et al.  Computational connectionism within neurons: a model of cytoskeletal automata subserving neural networks , 1990 .

[4]  P. Mangeat,et al.  An interaction between vinculin and talin , 1984, Nature.

[5]  L. Reichardt,et al.  Vinculin-deficient PC12 cell lines extend unstable lamellipodia and filopodia and have a reduced rate of neurite outgrowth , 1994, The Journal of cell biology.

[6]  M. Radmacher,et al.  From molecules to cells: imaging soft samples with the atomic force microscope. , 1992, Science.

[7]  D E Ingber,et al.  Cytoskeletal filament assembly and the control of cell spreading and function by extracellular matrix. , 1995, Journal of cell science.

[8]  A. Eisen,et al.  Integrin α2β1 (VLA-2) mediates reorganization and contraction of collagen matrices by human cells , 1991, Cell.

[9]  C. Turner,et al.  Paxillin: a new vinculin-binding protein present in focal adhesions , 1990, The Journal of cell biology.

[10]  K. Burridge,et al.  Disruption of the actin cytoskeleton after microinjection of proteolytic fragments of alpha-actinin , 1991, The Journal of cell biology.

[11]  W. Goldmann,et al.  Interaction of the 47-kDa talin fragment and the 32-kDa vinculin fragment with acidic phospholipids: a computer analysis. , 1995, Biophysical journal.

[12]  M. Hess.  High-pressure freeze fixation reveals novel features during ontogenesis of the vegetative cell in Ledebouria pollen: an ultrastructural and cytochemical study. , 1995, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[13]  R. Ezzell,et al.  Targeted disruption of vinculin genes in F9 and embryonic stem cells changes cell morphology, adhesion, and locomotion. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[14]  F. Doljanski,et al.  ON THE GENERATION OF FORM BY THE CONTINUOUS INTERACTIONS BETWEEN CELLS AND THEIR EXTRACELLULAR MATRIX , 1992, Biological reviews of the Cambridge Philosophical Society.

[15]  B. Jockusch,et al.  Intramolecular interactions in vinculin control α‐actinin binding to the vinculin head , 1994 .

[16]  H. Niwa,et al.  Efficient selection for high-expression transfectants with a novel eukaryotic vector. , 1991, Gene.

[17]  D. Critchley,et al.  The focal-adhesion vasodilator-stimulated phosphoprotein (VASP) binds to the proline-rich domain in vinculin. , 1996, The Biochemical journal.

[18]  P A Valberg,et al.  Magnetic particle motions within living cells. Physical theory and techniques. , 1987, Biophysical journal.

[19]  W. H. Goldmann,et al.  Viscoelasticity in wild-type and vinculin-deficient (5.51) mouse F9 embryonic carcinoma cells examined by atomic force microscopy and rheology. , 1996, Experimental cell research.

[20]  D. Ingber,et al.  Mechanotransduction across the cell surface and through the cytoskeleton , 1993 .

[21]  B. Jockusch,et al.  Characterization of an F-actin-binding domain in the cytoskeletal protein vinculin , 1994, The Journal of cell biology.

[22]  M. Rothkegel,et al.  The molecular architecture of focal adhesions. , 1995, Annual review of cell and developmental biology.

[23]  D E Ingber,et al.  Control of cytoskeletal mechanics by extracellular matrix, cell shape, and mechanical tension. , 1994, Biophysical journal.

[24]  Donald E. Ingber,et al.  How does extracellular matrix control capillary morphogenesis? , 1989, Cell.

[25]  V. Koteliansky,et al.  Interaction of iodinated vinculin, metavinculin and α‐actinin with cytoskeletal proteins , 1987 .

[26]  K. Matlin,et al.  Integrin expression and localization in normal MDCK cells and transformed MDCK cells lacking apical polarity. , 1994, Journal of cell science.

[27]  D A Lauffenburger,et al.  Integrin-cytoskeletal interactions in migrating fibroblasts are dynamic, asymmetric, and regulated , 1993, The Journal of cell biology.

[28]  K. Burridge,et al.  An interaction between alpha-actinin and the beta 1 integrin subunit in vitro , 1990, The Journal of cell biology.

[29]  A. Gilmore,et al.  Further characterisation of the talin-binding site in the cytoskeletal protein vinculin. , 1992, Journal of cell science.

[30]  Donald E. Ingber,et al.  CHAPTER 2 – Mechanochemical Transduction across Extracellular Matrix and through the Cytoskeleton , 1993 .

[31]  D. Ingber,et al.  Probing transmembrane mechanical coupling and cytomechanics using magnetic twisting cytometry. , 1995, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[32]  W. Goldmann,et al.  Motility of vinculin-deficient F9 embryonic carcinoma cells analyzed by video, laser confocal, and reflection interference contrast microscopy. , 1995, Experimental cell research.

[33]  C. Turner,et al.  Characterisation of the paxillin-binding site and the C-terminal focal adhesion targeting sequence in vinculin. , 1994, Journal of cell science.

[34]  John A. Frangos,et al.  Physical forces and the mammalian cell , 1993 .

[35]  H. Hansma,et al.  Biomolecular imaging with the atomic force microscope. , 1994, Annual review of biophysics and biomolecular structure.

[36]  M. M. Breuer.  Measuring the viscoelastic properties of aerosol shaving foams , 1984 .

[37]  S. Craig,et al.  F-actin binding site masked by the intramolecular association of vinculin head and tail domains , 1995, Nature.

[38]  V. Koteliansky,et al.  Organization of the human gene encoding the cytoskeletal protein vinculin and the sequence of the vinculin promoter. , 1993, The Journal of biological chemistry.

[39]  C. Coulson,et al.  Molecular Architecture , 1953, Nature.

[40]  P K Hansma,et al.  Measuring the viscoelastic properties of human platelets with the atomic force microscope. , 1996, Biophysical journal.

[41]  U. Walter,et al.  VASP interaction with vinculin: a recurring theme of interactions with proline‐rich motifs , 1996, FEBS letters.

[42]  R. Ezzell,et al.  Expression of chicken vinculin complements the adhesion-defective phenotype of a mutant mouse F9 embryonal carcinoma cell , 1993, The Journal of cell biology.

[43]  B. Geiger,et al.  Suppression of vinculin expression by antisense transfection confers changes in cell morphology, motility, and anchorage-dependent growth of 3T3 cells , 1993, The Journal of cell biology.

[44]  D E Ingber,et al.  Cellular tensegrity: exploring how mechanical changes in the cytoskeleton regulate cell growth, migration, and tissue pattern during morphogenesis. , 1994, International review of cytology.

[45]  S. Craig,et al.  An intramolecular association between the head and tail domains of vinculin modulates talin binding. , 1994, The Journal of biological chemistry.

[46]  D E Ingber,et al.  Vinculin promotes cell spreading by mechanically coupling integrins to the cytoskeleton. , 1997, Experimental cell research.

[47]  Z. Kam,et al.  Focal adhesion formation by F9 embryonal carcinoma cells after vinculin gene disruption. , 1995, Journal of cell science.

[48]  Yamamura Ken-ichi,et al.  Efficient selection for high-expression transfectants with a novel eukaryotic vector , 1991 .

[49]  Seema Singh,et al.  Scanning force microscopy of cells and membrane proteins , 1992, Photonics West - Lasers and Applications in Science and Engineering.